JP6458218B2 - High oleic oil - Google Patents

Description

  The present invention relates to extracted lipids containing high levels, for example 90% to 95% by weight oleic acid. The present invention also generally provides an oilseed such as safflower that can be used to produce genetically modified plants, specifically lipids. Further provided are methods for determining and selecting plant genotypes that can be used to produce lipids.

  Vegetable oil is an important source of dietary fat for humans, accounting for about 25% of caloric intake in developed countries (Broon et al., 1999). The world production of vegetable oil is at least about 110 million tonnes per year, of which 86% is used for human consumption. Almost all of these oils are obtained from oilseed crops such as soybeans, rapeseed, safflower, sunflower, cottonseed, and peanuts, or cultivated trees such as palm, olive, and coconut (Gunstone, 2001, Oil World Annual, 2004). The growing scientific understanding and social awareness of the effects of individual fatty acid components of edible oils in various aspects of human health has led to improved nutritional value while retaining the necessary functionality for various food applications The development of modified vegetable oils with These modifications require knowledge of the metabolic pathways for plant fatty acid synthesis and the genes that encode the enzymes for these pathways (Liu et al., 2002a, Thelen and Ohllogge, 2002).

  There is a great deal of interest in the nutritional effects of various fats and oils, specifically the effects of fat and oil components on cardiovascular disease, cancer, and various inflammatory conditions. High levels of cholesterol and saturated fatty acids in the diet are believed to increase the risk of heart disease, which reduces the consumption of cholesterol-rich saturated animal fats and reduces the use of unsaturated vegetable oils that do not contain cholesterol. Recommended nutritional advice was derived (Liu et al., 2002a).

  Although dietary intake of cholesterol present in animal fats can significantly increase the level of total cholesterol in the blood, we also see that fatty acids, including fats and oils themselves, can significantly affect serum cholesterol levels. It was issued. Of particular interest is the effect of dietary fatty acids on undesirable low density lipoprotein (LDL) and desirable high density lipoprotein (HDL) forms of cholesterol in the blood. In general, saturated fatty acids, particularly certain myristic acid (14: 0) and palmitic acid (16: 0), which are the major saturated fatty acids present in vegetable oils, increase serum LDL-cholesterol levels, resulting in heart Has undesirable properties that increase the risk of vascular disease (Zock et al., 1994, Hu et al., 1997). However, it is widely established that stearic acid (18: 0), the other major saturate present in vegetable oils, does not raise LDL-cholesterol and can actually lower total cholesterol (Bonanome and Grundy, 1988, Dougherty et al., 1995). Thus, stearic acid is generally regarded as at least neutral with respect to the risk of cardiovascular disease (Tholstrup, et al., 1994). On the other hand, unsaturated fatty acids such as monounsaturated oleic acid (18: 1) have the beneficial property of lowering LDL-cholesterol (Mensink and Katan, 1989, Roche and Gibney, 2000) and hence Reduce the risk of cardiovascular disease.

  Oils rich in oleic acid are also often in the form of fatty acid esters, lubricating oils, biofuels, fatty alcohol raw materials, plasticizers, waxes, metal stearates, emulsifiers, personal care products, soaps and detergents, interfaces Includes, but is not limited to, active agents, formulations, metalworking additives, softener raw materials, inks, clear soaps, PVC stabilizers, alkyd resins, and many other types of downstream oleochemical derivatives mediators, etc. Has many industrial uses that are not.

  Oil manufacturers and food manufacturers traditionally rely on hydrogenation to reduce the level of unsaturated fatty acids in the oil, thereby increasing oxidative stability in frying applications, and Provides solid fat for use in margarine and shortening. Hydrogenation is a chemical process that reduces the degree of unsaturation of an oil by converting carbon-carbon double bonds to carbon-carbon single bonds. Complete hydrolysis produces a fully saturated fat. However, the partial hydrogenation process results in increased levels of both saturated and monounsaturated fatty acids. Some of the monounsaturates formed during partial hydrogenation are not natural cis isomers but trans isomer forms (eg, elaidic acid, trans isomers of oleic acid) (Sebedio et al. 1994, Fernandez San Juan, 1995). In contrast to cis-unsaturated fatty acids, trans-fatty acids currently increase serum LDL cholesterol levels (Mensink and Katan, 1990, Noakes and Clifton, 1998) and lower serum HDL cholesterol (Zock and Katan, 1992). ) Above and is known to be as potent as palmitic acid and therefore contributes to an increased risk of cardiovascular disease (Ascherio and Willett, 1997). As a result of increased recognition of the nutritional inhibitory effects of trans fatty acids, currently the food industry avoids the use of hydrogenated oil, is nutritionally beneficial and can provide the necessary functionality without the need for hydrogenation And there is an increasing tendency to support oils, particularly those rich in either oleic acid where liquid oil is required or stearic acid where solid or semi-solid fats are preferred.

  There is a need for additional lipids and oils with high oleic acid content and its source.

  The inventors have created new lipid compositions and methods for producing these lipids.

In a first aspect, the present invention provides a lipid extracted from oil seeds, the lipid comprising triacylglycerol (TAG) consisting of fatty acids esterified to glycerol,
i) the fatty acid comprises palmitic acid and oleic acid,
ii) at least 95% by weight of the lipid is TAG;
iii) about 90% to about 95% by weight of the total fatty acid content of the lipid is oleic acid;
iv) Less than about 3.1% by weight of the total fatty acid content of the lipid is palmitic acid;
v) The lipid has an oleic unsaturation factor (ODP) of less than about 0.037 and / or a palmitic acid-linoleic acid-oleic acid value (PLO) of less than about 0.063.

In one embodiment, the lipid has one or more or all of the following characteristics.
a) about 90% to about 94%, or about 91% to about 94%, or about 91% to about 92%, or about 92%, or about 93% by weight of the total fatty acid content of the lipid; % Is oleic acid,
b) Less than about 3%, or less than about 2.75%, or less than about 2.5%, or about 3%, or about 2.75%, or about 2.% by weight of the total fatty acid content of the lipid. 5 wt%, or about 2.3 wt% is palmitic acid,
c) about 0.1% to about 3%, or about 2% to about 3%, or about 3%, or about 2% by weight of the total fatty acid content of the lipid is polyunsaturated fatty acid (PUFA) )
d) Less than about 3%, or less than about 2.5%, or less than about 2.25%, or about 3%, or about 2.5%, or about 2.% by weight of the total fatty acid content of the lipid. 25% by weight is linoleic acid,
e) less than about 1%, or less than about 0.5% by weight of the total fatty acid content of the lipid is α-linolenic acid (ALA);
f) about 0.5% to about 1% by weight of the total fatty acid content of the lipid is 18: 1Δ11.
g) The ODP of the total fatty acid content of the lipid is about 0.033 to about 0.01, or about 0.033 to about 0.016, or about 0.033 to about 0.023, or about 0.03. Or about 0.02, or about 0.01,
h) the PLO value of the total fatty acid content of the lipid is from about 0.020 to about 0.063, or from about 0.020 to about 0.055, or from about 0.020 to about 0.050, or from about 0.050 About 0.055, or about 0.063, or about 0.055, or about 0.050, or about 0.040, or about 0.030, or about 0.020,
i) about 90% to about 96%, or about 92% to about 96%, or about 93%, or about 94% by weight of the total fatty acid content of the lipid is a monounsaturated fatty acid;
j) the lipid has an oleic acid monounsaturation (OMP) of less than about 0.02, or less than about 0.015, or from about 0.005 to about 0.02.
k) lipids are in the form of refined oils,
l) Lipids are not hydrogenated.

In one embodiment,
1) About 91% to about 94% by weight of the total fatty acid content of the lipid is oleic acid,
2) Less than about 2.75% by weight of the total fatty acid content of the lipid is palmitic acid,
3) Less than about 3% by weight of the total fatty acid content of the lipid is linoleic acid,
4) α-linolenic acid is undetectable in the fatty acid content of the lipid,
5) The ODP of the fatty acid content of the lipid is from about 0.033 to about 0.023, or from about 0.033 to about 0.018.
6) The PLO value of the fatty acid content of the lipid is from about 0.020 to about 0.063.
7) About 96 wt%, or about 93 wt%, or about 94 wt% of the total fatty acid content of the lipid is monounsaturated fatty acid,
8) The lipid has an oleic acid monounsaturation rate (OMP) of less than about 0.02, or less than about 0.015, or from about 0.005 to about 0.02.

In a further embodiment,
1) About 91% to about 94% by weight of the total fatty acid content of the lipid is oleic acid,
2) Less than about 2.75% by weight of the total fatty acid content of the lipid is palmitic acid,
3) Less than about 3% by weight of the total fatty acid content of the lipid is linoleic acid,
4) α-linolenic acid is undetectable in the fatty acid content of the lipid,
5) about 96%, or about 93%, or about 94% by weight of the total fatty acid content of the lipid is a monounsaturated fatty acid, and 6) the lipid is less than about 0.02, or about 0.0. Oleic acid monounsaturation (OMP) of less than 015 or about 0.005 to about 0.02.

  In one embodiment, the PUFA is linoleic acid.

  In one embodiment, α-linolenic acid is undetectable in the fatty acid content of the lipid.

  In further embodiments, about 55% to about 80%, or about 60% to about 80%, or about 70% to about 80%, or at least about 60%, or at least about 70%, or about about the TAG content of the lipid 60%, or about 70%, or about 80% is triolein.

  In another embodiment, less than about 5%, or less than about 2%, or from about 0.1% to about 5% of the oleic acid content of the lipid is in the form of diacylglycerol (DAG).

  In further embodiments, the lipid is in the form of an oil and at least 90%, or at least 95%, at least about 98%, or from about 95% to about 98% by weight of the oil is lipid. .

  In a preferred embodiment, the oil seed is a non-photosynthetic oil seed. Examples of non-photosynthetic oil seeds include, but are not necessarily limited to, seeds from safflower, sunflower, cotton, or castor bean. In a preferred embodiment, the non-photosynthetic oil seed is a safflower seed.

  In one embodiment, the lipid further comprises one or more sterols.

  In a further embodiment, the lipid is in the form of an oil and comprises from about 5 mg sterol / g (oil) of oil, or from about 1.5 mg sterol / g (oil) to about 5 mg sterol / g (oil). .

In one embodiment, the lipid is
a) about 1.5% to about 4.5%, or about 2.3% to about 4.5% of the total sterol content is ergost-7-en-3β-ol,
b) about 0.5% to about 3%, or about 1.5% to about 3% of the total sterol content is triterpenoid alcohol,
c) from about 8.9% to about 20% of the total sterol content is Δ7-stigmasterol / stigmast-7-en-3β-ol, and d) from about 1.7% to about 6.% of the total sterol content. Including one or more or all of 1% is Δ7-avenasterol.

  In further embodiments, the lipid has been extracted from oil seeds having a volume of at least 1 liter and / or a weight of at least 1 kg and / or obtained from a field-grown plant.

  In one embodiment, the lipid is extracted from the oil seed by grinding and contains less than about 7% water by weight. In another embodiment, the lipid is a purified lipid (solvent extracted and purified) and comprises less than about 0.1 wt%, or less than about 0.05 wt% water.

  In another aspect, the present invention provides a composition comprising a first component that is a lipid of the present invention and a second component, preferably the composition mixes the lipid with the second component. Was generated by

  One skilled in the art will appreciate that the second component can be selected from a wide variety of compounds / compositions. In one example, the second component is a non-lipid substance such as an enzyme, a non-protein (non-enzyme) catalyst or chemical (eg, sodium hydroxide, methanol, or metal), or one or more ingredients of the diet.

The present invention also provides a process for producing oil, the process comprising:
i) obtaining an oil seed that is capable of producing a plant that comprises an oil and / or produces an oil seed comprising an oil, wherein the oil content of the oil seed is defined herein Obtaining oil seeds, which are lipids like
ii) extracting the oil from the oilseed, thereby producing the oil.

  In a preferred embodiment, the oil seed reduces the expression of the Δ12 desaturase (FAD2) gene in the developing oil seed as compared to a corresponding oil seed lacking the first exogenous polynucleotide. A first exogenous polynucleotide encoding a first silencing RNA that can be operably linked to a promoter that directs expression of the polynucleotide in a developing oil seed .

In another aspect, the present invention provides a process for producing oil, the process comprising:
i) seeds containing triacylglycerols (TAG) comprising fatty acids esterified to glycerol and / or triacylglycerols (TAG) comprising fatty acids esterified to glycerol. Can produce plants to produce,
a) the fatty acid comprises palmitic acid and oleic acid,
b) at least 95% by weight of the oil content of the seed is TAG;
c) about 75% to about 95% by weight of total fatty acids in the oil content of the seed is oleic acid;
d) Less than about 5.1% by weight of the total fatty acids in the oil content of the seed is palmitic acid,
e) safflower seeds wherein the oil content of the seed has an oleic unsaturation rate (ODP) of less than about 0.17 and / or a palmitic acid-linoleic acid-oleic acid (PLO) value of less than about 0.26 And getting
ii) extracting oil from safflower seeds, thereby producing oil,
A first siren capable of reducing expression of a Δ12 desaturase (FAD2) gene in a developing safflower seed as compared to a corresponding safflower seed lacking a first exogenous polynucleotide A first exogenous polynucleotide that encodes the singing RNA is operably linked to a promoter that directs expression of the polynucleotide in the developing safflower seed.

  In one embodiment, the oilseed or safflower seed is palmitoyl in the developing oilseed or safflower seed as compared to the corresponding oilseed or safflower seed lacking the second exogenous polynucleotide. A second exogenous polynucleotide encoding a second silencing RNA capable of reducing the expression of the ACP thioesterase (FATB) gene, wherein the second exogenous polynucleotide is a developing oil seed Alternatively, it is operably linked to a promoter that induces expression of the polynucleotide in safflower seeds.

  In another embodiment, the oilseed or safflower seed is in a developing oilseed or safflower seed compared to a corresponding oilseed or safflower seed lacking a third exogenous polynucleotide. A third exogenous polynucleotide encoding a third silencing RNA capable of reducing expression of a plastid ω6 fatty acid desaturase (FAD6) gene, wherein the third exogenous polynucleotide comprises a developing oil It is operably linked to a promoter that induces expression of the polynucleotide in seed or safflower seed.

In one embodiment,
1) The FAD2 gene is one or more or each of a CtFAD2-1 gene, a CtFAD2-2 gene, and a CtFAD2-10 gene, preferably a CtFAD2-1 gene and / or a CtFAD2-2 gene, and / or 2 ) The FATB gene is a CtFATB-3 gene and / or 3) The FAD6 gene is a CtFAD6 gene.

In one embodiment, the oil content of safflower seed has one or more or all of the following characteristics.
a) from about 80% to about 94%, or from about 85% to about 94%, or from about 90% to about 94%, or from about 91% to about 91% by weight of the total fatty acids of the seed oil content 94 wt%, or about 91 wt% to about 92 wt%, or about 92 wt%, or about 93 wt% is oleic acid,
b) less than about 5%, or less than about 4%, or less than about 3%, or less than about 2.75%, or less than about 2.5%, or less than about 5% by weight of the total fatty acids of the oil content of the seed; About 3%, or about 2.75%, or about 2.5% by weight is palmitic acid,
c) from about 0.1% to about 15%, or from about 0.1% to about 10%, or from about 0.1% to about 7.5% by weight of the total fatty acids of the seed oil; or About 0.1% to about 5%, or about 0.1% to about 3%, or about 2% to about 3%, or about 3%, or about 2% by weight A polyunsaturated fatty acid (PUFA),
d) Less than about 15%, or less than about 10%, or less than about 5%, or less than about 3%, or less than about 2.5%, or about 2.25% of the total fatty acids of the seed oil. Less than wt%, or about 3 wt%, or about 2.5 wt%, or about 2.25 wt% is linoleic acid (LA).
e) about 80% to about 96%, or about 90% to about 96%, or about 92% to about 96%, or about 93%, or about 94% by weight of the total fatty acid content of the lipid. % Is monounsaturated fatty acid,
f) The lipid is less than about 0.05, or less than about 0.02, or less than about 0.015, or from about 0.005 to about 0.05, or from about 0.005 to about 0.02 monooleic acid. Having a valence unsaturation (OMP),
g) The ODP of the seed oil content is from about 0.17 to about 0.01, or from about 0.15 to about 0.01, or from about 0.1 to about 0.01, or from about 0.075 to about 0.01, or about 0.050 to about 0.01, or about 0.033 to about 0.01, or about 0.033 to about 0.016, or about 0.033 to about 0.023 Or about 0.03, or about 0.02, or about 0.01,
h) The PLO value of the oil content of the seed is about 0.20 to about 0.026, or about 0.020 to about 0.2, or about 0.020 to about 0.15, or about 0.020 to About 0.1, or about 0.020 to about 0.075, or about 0.050 to about 0.055, or about 0.05, or about 0.040, or about 0.030, or about 0.020 It is.

  In one embodiment, the step of extracting oil comprises grinding oil seeds or safflower seeds.

  In yet another embodiment, the process further comprises purifying the oil extracted from the oilseed or safflower seed, the purification step degumming, deodorizing, decolorizing, drying, and drying the extracted oil. One or more of the group consisting of: / or fractionating and / or removing at least some, preferably substantially all, waxes and / or wax esters from the extracted oil, or Includes everything.

In another embodiment, the present invention comprises triacylglycerol (TAG), wherein the oil content comprises a fatty acid esterified to glycerol, and / or a triacylglycerol, wherein the oil content comprises a fatty acid esterified to glycerol. Providing an oilseed capable of producing a plant that produces an oilseed comprising (TAG),
i) the fatty acid comprises palmitic acid and oleic acid,
ii) at least 95% by weight of the oil content of the oil seed is TAG;
iii) about 90% to about 95% by weight of the total fatty acids of the oil content of the oil seed is oleic acid;
iv) Less than about 3.1% by weight of the total fatty acids in the oil-containing oil seeds is palmitic acid;
v) The oil content of the oil seed has an oleic unsaturation rate (ODP) of less than about 0.037 and / or a palmitic acid-linoleic acid-oleic acid (PLO) value of less than about 0.063.

  In one embodiment, the oil seeds are non-photosynthetic oil seeds, preferably seeds from safflower, sunflower, cotton, or castor bean.

  In another embodiment, the oil seed allows expression of the Δ12 desaturase (FAD2) gene in the developing oil seed as compared to a corresponding oil seed lacking an exogenous polynucleotide. Or a first exogenous polynucleotide that encodes a first silencing RNA that reduces, wherein the first exogenous polynucleotide is operable to a promoter that induces expression of the polynucleotide in a developing oil seed Connected to

In yet a further aspect, the present invention comprises triacylglycerol (TAG), wherein the oil content comprises a fatty acid esterified to glycerol, and / or a triacylglycerol, wherein the oil content comprises a fatty acid esterified to glycerol. Providing safflower seeds capable of producing plants that produce seeds comprising (TAG),
i) the fatty acid comprises palmitic acid and oleic acid,
ii) at least 95% by weight of the oil content of the seed is TAG;
iii) about 75% to about 95% by weight of the total fatty acids of the oil content of the seed is oleic acid;
iv) less than about 5.1% by weight of the total fatty acids of the oil content of the seed is palmitic acid;
v) the oil content of the seed has an oleic unsaturation rate (ODP) of less than about 0.17 and / or a palmitic acid-linoleic acid-oleic acid (PLO) value of less than about 0.26;
The first siren capable of reducing the expression of the Δ12 desaturase (FAD2) gene in the developing safflower seed as compared to a corresponding safflower seed lacking the first exogenous polynucleotide. A first exogenous polynucleotide that encodes a singing RNA, wherein the first exogenous polynucleotide is operably linked to a promoter that induces expression of the polynucleotide in the developing safflower seed.

In one embodiment, the oil content of safflower seeds has one or more of the following characteristics.
a) from about 80% to about 94%, or from about 85% to about 94%, or from about 90% to about 94%, or from about 91% to about 91% by weight of the total fatty acids of the seed oil content 94 wt%, or about 91 wt% to about 92 wt%, or about 92 wt%, or about 93 wt% is oleic acid,
b) less than about 5%, or less than about 4%, or less than about 3%, or less than about 2.75%, or less than about 2.5%, or less than about 5% by weight of the total fatty acids of the oil content of the seed; About 3%, or about 2.75%, or about 2.5% by weight is palmitic acid,
c) from about 0.1% to about 15%, or from about 0.1% to about 10%, or from about 0.1% to about 7.5% by weight of the total fatty acids of the seed oil content Or about 0.1 wt% to about 5 wt%, or about 0.1 wt% to about 3 wt%, or about 2 wt% to about 3 wt%, or about 3 wt%, or about 2 wt% A polyunsaturated fatty acid (PUFA),
d) Less than about 15%, or less than about 10%, or less than about 5%, or less than about 3%, or less than about 2.5%, or about 2.25% of the total fatty acids of the seed oil. Less than wt%, or about 3 wt%, or about 2.5 wt%, or about 2.25 wt% is linoleic acid (LA).
e) about 80% to about 96%, or about 90% to about 96%, or about 92% to about 96%, or about 93%, or about 94% by weight of the total fatty acid content of the lipid. % Is monounsaturated fatty acid,
f) less than about 0.05, or less than about 0.02, or less than about 0.015, or about 0.005 to about 0.05, or about 0.005 to about 0.02 oleic acid mono Having a valence unsaturation (OMP),
g) The ODP of the seed oil content is from about 0.17 to about 0.01, or from about 0.15 to about 0.01, or from about 0.1 to about 0.01, or from about 0.075 to about 0.01, or about 0.050 to about 0.01, or about 0.033 to about 0.01, or about 0.033 to about 0.016, or about 0.033 to about 0.023 Or about 0.03, or about 0.02, or about 0.01,
h) The PLO value of the oil content of the seed is about 0.020 to about 0.26, or about 0.020 to about 0.2, or about 0.020 to about 0.15, or about 0.020 to About 0.1, or about 0.020 to about 0.075, or about 0.050 to about 0.055, or about 0.050, or about 0.040, or about 0.030, or about 0.020 It is.

  In a further embodiment, the oil content of the oil seed or safflower seed is a lipid further characterized by one or more of the above-described characteristics.

  In another embodiment, the oil seed or safflower seed is in a developing oil seed or safflower seed as compared to a corresponding oil seed or safflower seed lacking the second exogenous polynucleotide. A second exogenous polynucleotide encoding a second silencing RNA capable of reducing the expression of a palmitoyl-ACP thioesterase (FATB) gene, wherein the second exogenous polynucleotide comprises a developing oil It is operably linked to a promoter that induces expression of the polynucleotide in seed or safflower seed.

  In a further embodiment, the oil seed or safflower seed is pigmented in the developing oil seed or safflower seed as compared to a corresponding oil seed or safflower seed lacking the third exogenous polynucleotide. A third exogenous polynucleotide capable of reducing the expression of a body ω6 fatty acid desaturase (FAD6) gene, wherein the third exogenous polynucleotide is capable of reducing the expression of the polynucleotide in a developing oilseed or safflower seed. It is operably linked to an inducible promoter.

  In another embodiment, the first silencing RNA reduces expression of more than one exogenous gene encoding FAD2 in developing oil seeds or safflower seeds, and / or a second silencing RNA. Reduces the expression of more than one exogenous gene encoding FATB in developing oil or safflower seeds.

  In still further embodiments, the first exogenous polynucleotide and either or both of the second exogenous polynucleotide and the third exogenous polynucleotide, optionally in the first DNA molecule, Covalently binds to a linking DNA sequence between the second, and / or third exogenous polynucleotide.

  In another embodiment, the first exogenous polynucleotide and either or both of the second exogenous polynucleotide and the third exogenous polynucleotide are under the control of a single promoter, and therefore When the first exogenous polynucleotide and the second exogenous polynucleotide and / or the third exogenous polynucleotide are transcribed in the developing oil seed or safflower seed, the first silencing RNA and the second The silencing RNA and / or the third silencing RNA are covalently linked as part of a single RNA transcript.

  In another embodiment, the oil seed or safflower seed comprises a single transfer DNA integrated into the genome of the oil seed or safflower seed, the single transfer DNA comprising a first exogenous polynucleotide, and Includes either or both of the second exogenous polynucleotide and the third exogenous polynucleotide.

  Preferably, the oil seed or safflower seed is homozygous for the transfer DNA.

  In one embodiment, the first silencing RNA, the second silencing RNA, and the third silencing RNA are each independently an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, a microRNA, and a second Selected from the group consisting of single stranded RNA.

  In a further embodiment, any one or more, preferably all, of the promoters are seed-specific, preferably selectively expressed in the developing oilseed or safflower seed embryo.

  In another embodiment, the oilseed or safflower seed comprises one or more mutants in one or more FAD2 genes, and the mutant (s) comprises the mutant (s). The activity of one or more FAD2 genes is reduced in the developing oil seed or safflower seed compared to the corresponding oil seed or safflower seed lacking.

  In another embodiment, the oil seed or safflower seed comprises a mutant of the FAD2 gene as compared to the wild type FAD2 gene in the corresponding oil seed or safflower seed, the mutation being deleted, inserted , Inversion, frameshift, early translation stop codon, or one or more non-conservative amino acid substitutions.

  In a further embodiment, the mutation is a null mutation in the FAD2 gene.

  In another embodiment, at least one of the mutants is developing in which the mutant (s) are deleted from any other FAD2 gene in the developing oilseed or safflower seeds Within the FAD2 gene, which encodes additional FAD2 activity in middle oilseed or safflower seeds.

  In another embodiment, the seed is a safflower seed and the FAD2 gene is a CtFAD2-1 gene. In this embodiment, the first silencing RNA can at least reduce the expression of the CtFAD2-2 gene.

  In another embodiment, the seed is a safflower seed comprising the ol allele of the CtFAD2-1 gene, the ol1 allele of the CtFAD2-1 gene, or both alleles. In one embodiment, the ol allele or ol1 allele of the CtFAD2-1 gene is present in a homozygous state.

  In another embodiment, FAD2 protein is not detectable in oil seeds or safflower seeds.

  In another embodiment, the seed is a safflower seed and the first silencing RNA reduces the expression of both the CtFAD2-1 and CtFAD2-2 genes.

In another embodiment, the seed is a safflower seed,
1) The FAD2 gene is one or more of a CtFAD2-1 gene, a CtFAD2-2 gene, and a CtFAD2-10 gene, preferably a CtFAD2-1 gene and / or a CtFAD2-2 gene, and / or 2 ) FATB gene is CtFATB-3 gene and / or 3) FAD6 gene is CtFAD6 gene.

  Also provided are oil seed plants or safflower plants that are capable of producing the seeds of the present invention.

  In one embodiment, the plant is transgenic, homozygous for each exogenous polynucleotide, and / or the first exogenous polynucleotide, and the second exogenous polynucleotide or the third exogenous polynucleotide. Includes either or both of the polynucleotides.

  In another aspect, the invention provides a sequence provided in any one of SEQ ID NOs: 27-37, 44, 45, or 48, a biologically active fragment thereof, or SEQ ID NOs: 27-37, Provided are substantially purified and / or recombinant polypeptides comprising amino acids having an amino acid sequence that is at least 40% identical to any one or more of 44, 45, or 48.

  In one embodiment, a polypeptide that is an enzyme that modifies fatty acids, preferably oleate Δ12 desaturase, Δ12-acetylenase, palmitoleate Δ12 desaturase, or palmitoyl-ACP thioesterase (FATB).

In a further aspect, the present invention provides isolated and / or exogenous polynucleotides,
i) a nucleotide having the sequence provided in any one of SEQ ID NOs: 1-25, 40-43, 46, or 47;
ii) a nucleotide having a sequence encoding a polypeptide of the invention,
iii) a nucleotide that hybridizes to a sequence provided in any one of SEQ ID NOs: 1-25, 40-43, 46, or 47;
iv) when expressed in the seed of an oilseed plant, contains one or more of nucleotides having a sequence that reduces the expression of a gene encoding at least one polypeptide of the invention.

  In a particularly preferred embodiment, the polynucleotide comprises a nucleotide nucleotide having a sequence that, when expressed in the seed of an oilseed plant, reduces the expression of a gene encoding at least one polypeptide of the invention.

  In one embodiment, the polynucleotide of part iv) comprises a sequence of nucleotides provided in any one or more of SEQ ID NOs: 49-51 (thymine (T) is uracil (U)).

  In one embodiment, the iv) part polynucleotide is selected from an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, a microRNA, a double stranded RNA (dsRNA) molecule, or a processed RNA product thereof. .

  In further embodiments, the polynucleotide is a dsRNA molecule, or a processed RNA product thereof, and SEQ ID NOs: 1-25, 40-43, 46, 47, or 49-51 (thymine (T) is uracil (U ) Is at least 19 contiguous nucleotides that are at least 95% identical to one or more complements.

  In another embodiment, the dsRNA molecule is a microRNA (miRNA) precursor and / or the processed RNA product is a miRNA.

  In still further embodiments, the polynucleotide is transcribed in developing oil or safflower seeds under the control of a single promoter, and complementary sense and antisense that allow dsRNA molecules to hybridize to each other. It contains sequences and is linked by single stranded RNA regions.

In still further embodiments, when present in a developing safflower seed, the polynucleotide is
i) reducing the expression of an endogenous gene encoding oleate Δ12 desaturase (FAD2) in the developing seed, wherein FAD2 is any one or more of SEQ ID NOs: 27, 28, or 36, preferably Having an amino acid sequence provided in at least one or both of SEQ ID NOs: 27 and 28;
ii) reducing the expression of an endogenous gene encoding palmitoyl-ACP thioesterase (FATB) in the developing seed, wherein FATB has the amino acid sequence provided in SEQ ID NO: 45, and / or iii) developing Of the gene encoding ω6 fatty acid desaturase (FAD6) in the seeds of FAD6, FAD6 has the amino acid sequence provided in SEQ ID NO: 48.

  Also provided is a chimeric vector comprising a polynucleotide of the invention operably linked to a promoter.

  In one embodiment, the promoter is functional in oil seeds or is a seed-specific promoter, and is preferably selectively expressed in developing oil seed embryos.

  In another aspect, the invention provides a recombinant cell comprising an exogenous polynucleotide of the invention and / or a vector of the invention.

  The cells can be any cell type, including but not limited to bacterial cells, yeast cells, or plant cells.

  Preferably the cell is a plant cell, preferably a plant seed cell. More preferably, the plant cell is an oil seed plant cell. Even more preferably, the plant cell is a non-photosynthetic seed cell, preferably a safflower, cotton or castor seed cell.

  In another aspect, the present invention provides a transgenic non-human organism comprising one or more of the polynucleotides of the present invention, the vectors of the present invention, and the cells of the present invention.

  Preferably, the transgenic non-human organism is a plant. More preferably, an oilseed plant.

  In yet another aspect, the present invention provides a method for producing a cell of the present invention, the method comprising introducing a polynucleotide of the present invention and / or a vector of the present invention into the cell.

  Also provided is the use of a polynucleotide of the invention and / or a vector of the invention to produce a recombinant cell.

In another aspect, the invention provides a transgenic oilseed plant that produces the seeds of the invention, or a method of producing the seeds, the method comprising:
i) introducing at least one polynucleotide of the invention and / or at least one vector of the invention into cells of an oilseed plant;
ii) regenerating a transgenic plant from the cells;
iii) optionally generating one or more progeny plants or seeds thereof from the transgenic plant;
Thereby producing a transgenic oilseed plant or its seeds.

  In one embodiment, the seed is a safflower seed of the invention.

In a further embodiment, the one or more progeny plants or their seeds are
i) a first exogenous polynucleotide, and / or ii) a second exogenous polynucleotide, and / or iii) a third exogenous polynucleotide,
Preferably, all three exogenous polynucleotides are included.

  In a further embodiment, the one or more progeny plants or seeds thereof comprise one or more mutants as defined above.

In yet a further aspect, the present invention provides a method of obtaining an oilseed plant, the method comprising:
i) a first parent oilseed plant comprising the first polynucleotide of the present invention or the first vector of the present invention, the first parent comprising the second polynucleotide of the present invention or the first vector of the present invention; Crossing with two parent oilseed plants,
ii) screening progeny plants from the cross for the presence of both polynucleotides or both vectors;
iii) Increasing the ratio of oleic acid in the oil content of plant seeds, comprising both (a) a first polynucleotide or first vector, and (b) a second polynucleotide or second vector And selecting progeny plants further having a reduced ratio of palmitic acid.

  In a further aspect, the present invention is a method for determining the genotype of a safflower plant, the method comprising detecting a nucleic acid molecule of the plant, the nucleic acid molecule comprising CtFAD2-1, CtFAD2- of the safflower plant. 2 or one or more of CtFAD2-10 genes, preferably linked to at least one or both of CtFAD2-1 and CtFAD2-2 genes, or at least CtFAD2-1 genes, and / or at least Including some of them.

  In one embodiment, the method comprises determining the level of expression and / or sequence of one or more of the plant CtFAD2-1, CtFAD2-2, or CtFAD2-10 genes.

In one embodiment, the method comprises:
i) hybridizing a second nucleic acid molecule to the nucleic acid molecule of the plant;
ii) optionally hybridizing at least one other nucleic acid molecule to the nucleic acid molecule of the plant;
iii) detecting the product of the hybridizing step (s) or the absence of the product from the hybridizing step (s).

  In still further embodiments, the second nucleic acid molecule is used as a primer for reverse transcription or replication of at least a portion of a plant nucleic acid molecule.

  Nucleic acids use various well-known techniques including, but not limited to, restriction fragment length polymorphism analysis, amplified fragment length polymorphism analysis, microsatellite amplification, nucleic acid sequencing, and / or nucleic acid amplification, etc. Can be detected.

  In one embodiment, the method detects the presence or absence of an allele of the CtFAD2-1 gene, preferably the ol allele.

In a further aspect, the present invention provides a method for selecting a safflower plant from a safflower plant population, the method comprising:
i) using the method of the invention to determine the genotype of the plant population, wherein the plant population is obtained from a cross between two plants, at least one of which is CtFAD2- 1, CtFAD2-2, or CtFAD2-10 gene, preferably at least one or both of CtFAD2-1 and CtFAD2-2 genes, or at least an allele of CtFAD2-1 gene, and development of seeds of the plant Determining the genotype of the plant population that results in a reduced level of Δ12 desaturase activity compared to the corresponding seed of a safflower plant lacking the allele;
ii) selecting a safflower plant based on the presence or absence of the allele.

In a further aspect, the present invention provides a method for introducing an allele of a CtFAD2-1, CtFAD2-2, or CtFAD2-10 gene into a safflower plant lacking the allele, the method comprising:
i) crossing a first parent safflower plant with a second parent safflower plant, wherein said second plant comprises said allele of the CtFAD2-1, CtFAD2-2, or CtFAD2-10 gene Including, mating,
ii) to generate the progeny of the mating of step i) with a plant of the same genotype as the first parent plant and a plant comprising the majority of the first parent genotype but containing the allele Back mating a sufficient number of times,
The allele results in a decrease in the level of Δ12 desaturase activity compared to the corresponding seed of the safflower plant lacking the allele during the development of the plant seed, and the progeny plant is a method of the present invention. Is used to determine the genotype for the presence or absence of the allele.

  Also provided is a transgenic plant, or progeny plant thereof, or seed thereof produced using the method of the present invention.

In a further aspect, the present invention provides a method for producing seed, the method comprising:
a) growing the plants of the present invention in the region, preferably as part of at least 1000 such plant populations;
b) harvesting the seeds.

  In a still further aspect, the present invention relates to an oil seed or safflower seed of the present invention, a plant of the present invention or a part thereof, a cell of the present invention, and / or a non-human transgenic organism of the present invention or one of the same. Provides an oil obtained or obtainable by one or more of the processes of the present invention.

  In another aspect, the invention provides a lipid of the invention, an oil seed or safflower seed of the invention, a polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, or the invention. A composition comprising one or more of the following oils and one or more acceptable carriers.

  Also, for the production of industrial products, the lipids of the invention, the compositions of the invention, the processes of the invention, the oil seeds or safflower seeds of the invention, the plants of the invention or parts thereof, the hosts of the invention There is provided the use of one or more of a cell, a non-human transgenic organism of the invention or part thereof, or an oil of the invention.

In another aspect, the present invention provides a process for producing an industrial product, the process comprising:
i) lipids of the invention, compositions of the invention, oil seeds or safflower seeds of the invention, plants of the invention or parts thereof, host cells of the invention, non-human transgenic organisms of the invention or parts thereof Or obtaining one or more of the oils of the present invention;
ii) The lipid of the present invention, the composition of the present invention, the oil seed or safflower seed of the present invention, the plant of the present invention or a part thereof, the host cell of the present invention, the non-human transgenic of the present invention in step i) Optionally physically treating one or more of the organism or part thereof, or the oil of the present invention;
ii) a lipid of the invention, or one or more lipids of the composition of the invention, an oil seed or safflower seed of the invention, a plant of the invention or part thereof, a host cell of the invention, a A non-human transgenic organism or part thereof, or an oil of the invention, or at least a part of the physically treated product of step ii), is subjected to thermal, chemical or enzymatic methods, or any combination thereof Converting to an industrial product by applying to the lipid;
iii) recovering industrial products,
Thereby producing an industrial product.

In yet a further aspect, the present invention provides a method of producing fuel, the method comprising:
i) a lipid of the invention, one or more lipids of the invention, an oil seed or safflower seed of the invention, a plant of the invention or part thereof, a host cell of the invention, a non-human transgenic organism of the invention or Reacting one or more of a portion thereof, or an oil of the invention, with an alcohol, optionally in the presence of a catalyst, to form an alkyl ester;
ii) optionally mixing the alkyl ester with petroleum fuel.

  In one embodiment, the alkyl ester is a methyl ester.

  In a further aspect, the present invention provides a method for producing feed, the method comprising a lipid of the present invention, a composition of the present invention, an oil seed or safflower seed of the present invention, a plant of the present invention or a part thereof. Mixing one or more of the host cells according to the invention, the non-human transgenic organism of the invention or a part thereof, or the oil of the invention with at least one other plant component.

  The lipid of the present invention, the composition of the present invention, the oil seed or safflower seed of the present invention, the plant of the present invention or a part thereof, the host cell according to the present invention, the non-human transgenic organism of the present invention or the same Also provided are feeds, cosmetics, or chemicals comprising one or more of a portion or one of the oils of the present invention.

  In a still further aspect, the lipid of the invention, one or more lipids of the composition of the invention, the process of the invention, the oil seed or safflower seed of the invention, the plant of the invention or part thereof, the invention Products produced from or using one or more of the present host cells, non-human transgenic organisms of the invention or parts thereof, or oils of the invention are provided.

  Any embodiment of this specification shall be construed as being modified and applied to any other embodiment unless specifically stated otherwise.

  The present invention is not to be limited in scope by the specific embodiments described herein, but is intended to be exemplary only. Functionally equivalent products, compositions, and methods as described herein are clearly within the scope of the invention.

  Throughout this specification, references to a single step, composition, step group, or composition group, unless specifically stated otherwise, or unless the context requires otherwise, refer to one and more ( Ie, one or more) such steps, compositions, groups of steps or groups of compositions.

  The invention will now be described by the following non-limiting examples and with reference to the accompanying drawings.

Phylogenetic comparison of amino acid sequences encoded by safflower FAD2-like gene family and FAD2-like enzymes branched from other plants. The phylogenetic tree shown was created by use of the vector NTI (invitrogen). FAD2 desaturase (DES), hydroxylase (OH), epoxygenase (EPOX), acetylenase (ACET), and conjugase (CONJ) were included in the alignment. The GeneBank accession numbers of the amino acid sequences shown in the phylogenetic tree are: coCONJ, AAK26632.1, caACET, ABC00769.1, cpEPOX, CAA76156.1, haACET, ABC596844.1, dsACET, AAO38036.1, dcACET, AA38033. , HhACET, AAO38031.1, haDES-2, AAL68982.1, haDES-3, AAL68983.1, luDES, ACF49507, haDES-1, AAL68982.1, ntDES, AAT722296, oeDES, AAW63040, siDES, AAF80560.1 GhDES-1, CAA65744.1, rcOH, AAC490010.1, atDES, AAM61113.1, fOH: DES, AAC32755.1, plOH, ABQ01458.1, ghDES-4, AAQ16653.1, ghDES-2, CAA71199.1. (Co, Calendula officinalis, ca, Crepis alpine, cp, Crepis palaestina, ha, Helianthus annuus, ds, Dimorphotheca sinuate, dc, Daucus carota, hh, Hedera helix, lu, Linum usitatissimum, nt, Nicotiana tabacum, oe, Olea europaea , Si, Sesamum indicum, rc, Ricinus communis, at, Arabidopsis thaliana, pf, Physaria fendelii, pl, Physiological Lindheimeri). Southern blot hybridization analysis of CtFAD2-like genomic structure in safflower genotype SU. Genomic DNA was digested with 8 different restriction enzymes prior to separation on an agarose gel. These enzymes were AccI (lane 1), BglII (2), BamHI (3), EcoRI (4), EcoRV (5), HindIII (6), XbaI (7), and XhoI (8). The blot was probed with the entire radiolabeled coding region of CtFAD2-6 and washed under low stringency conditions. GC-MS fatty acid analysis of fatty acid composition from yeast expressing CtFAD2-1 (B), CtFAD2-2 (C), CtFAD2-9 (D), CtFAD2-10 (E), and CtFAD2-11 (F) . GC trace of a mixture of empty vector (A) and C18: 2 isomer (G). N. GC-MS fatty acid analysis of fatty acid composition after CtFAD2-11 transiently expressed in Benthamiana leaves. RT-qPCR expression analysis of safflower CtFAD2 gene. Of the CtFAD2-1 allele from wild-type SU and three high oleic acid genotypes, namely S-317, CW99-OL, and Lesaf496, showing a nucleotide deletion in the middle of the CtFAD2-1 coding region in the mutant Nucleotide comparison of regions. DNA sequence comparison of CtFAD2-1 5'UTR intron in wild type variant SU and high oleic acid genotype S-317. The DNA sequence in the box was used to design a complete PCR marker for high oleic acid specific and wild type specific PCR products. Real-time q-PCR analysis of CtFAD2-1 and CtFAD2-2 mRNA levels in developing embryos at three developmental stages, early (7 DPA), intermediate (15 DPA), and late (20 DPA). Safflower varieties include wild-type SU and three high oleic acid varieties: S-317, CW99-OL, and Lesaf496. Real-time qPCR analysis of the CtFatB gene in leaves, roots, and developing embryos of safflower variant SU. Em-1 (early), Em-2 (medium), and Em-3 (late). Phylogenetic tree showing the phylogenetic relationship between the safflower FAD6 sequence and the FAD6 plastid Δ12 desaturase identified in higher plants. Jatropha curcas (EU1068889), Olea europaea (AY733075), Populus trichocarpa (EF147523), Arabidopsis aurida 39 sophia) (EF524189), glycine max (AK243928), brassica napus (L29214), portulaca oleracea (EU376530), arachis hippoge (Aracis hapiroco) Ginkgo biloba) (HQ694456). S317 vs. S317 + 603.9 diacylglycerol (DAG) composition (mol%) by LC-MS analysis of a single seed. Triacylglycerol (TAG) composition (mol%) of S317 vs. S317 + 603.9 by LC-MS analysis of a single seed. Oleic acid content of safflower varieties under field conditions at Narrabri in Australian summer 2011/2012. (A) Basic plant sterol structure with cyclic side chain numbering. (B) Chemical structure of some plant sterols.

Description of Sequence Listing SEQ ID NO: 1-cDNA encoding safflower FAD2-1.
SEQ ID NO: 2- cDNA encoding safflower FAD2-2.
SEQ ID NO: 3- cDNA encoding safflower FAD2-3.
SEQ ID NO: 4- cDNA encoding safflower FAD2-4.
SEQ ID NO: 5- cDNA encoding safflower FAD2-5.
SEQ ID NO: 6- cDNA encoding safflower FAD2-6.
SEQ ID NO: 7-cDNA encoding safflower FAD2-7.
SEQ ID NO: 8-cDNA encoding safflower FAD2-8.
SEQ ID NO: 9-cDNA encoding safflower FAD2-9.
SEQ ID NO: 10-cDNA encoding safflower FAD2-10.
SEQ ID NO: 11—cDNA encoding safflower FAD2-11.
SEQ ID NO: 12—Open reading frame encoding safflower FAD2-1.
SEQ ID NO: 13—Open reading frame encoding safflower FAD2-2.
SEQ ID NO: 14—Open reading frame encoding safflower FAD2-3.
SEQ ID NO: 15—Open reading frame encoding safflower FAD2-4.
SEQ ID NO: 16—Open reading frame encoding safflower FAD2-5.
SEQ ID NO: 17—Open reading frame encoding safflower FAD2-6.
SEQ ID NO: 18—Open reading frame encoding safflower FAD2-7.
SEQ ID NO: 19—Open reading frame encoding safflower FAD2-8.
SEQ ID NO: 20—Open reading frame encoding safflower FAD2-9.
SEQ ID NO: 21—Open reading frame encoding safflower FAD2-10.
SEQ ID NO: 22—Open reading frame encoding safflower FAD2-11.
SEQ ID NO: 23—Intron sequence of safflower FAD2-1 gene.
SEQ ID NO: 24—Intron sequence of safflower FAD2-2 gene.
SEQ ID NO: 25—Intron sequence of safflower FAD2-10 gene.
SEQ ID NO: 26—cDNA encoding truncated safflower FAD2-1 (HO mutant).
SEQ ID NO: 27-Safflower FAD2-1.
SEQ ID NO: 28-Safflower FAD2-2.
SEQ ID NO: 29-Safflower FAD2-3.
SEQ ID NO: 30-Safflower FAD2-4.
SEQ ID NO: 31-Safflower FAD2-5.
SEQ ID NO: 32-Safflower FAD2-6.
SEQ ID NO: 33-Safflower FAD2-7.
SEQ ID NO: 34-Safflower FAD2-8.
SEQ ID NO: 35-Safflower FAD2-9.
SEQ ID NO: 36-Safflower FAD2-10.
SEQ ID NO: 37-Safflower FAD2-11.
SEQ ID NO: 38-cleaved safflower FAD2-1 (HO mutant).
SEQ ID NO: 39-cDNA for safflower FATB-1.
SEQ ID NO: 40-cDNA encoding safflower FATB-2.
SEQ ID NO: 41-cDNA encoding safflower FATB-3.
SEQ ID NO: 42—Open reading frame encoding safflower FATB-2.
SEQ ID NO: 43—Open reading frame encoding safflower FATB-3.
SEQ ID NO: 44-Safflower FATB-2.
SEQ ID NO: 45-Safflower FATB-3.
SEQ ID NO: 46—cDNA encoding safflower FAD6.
SEQ ID NO: 47—Open reading frame encoding safflower FAD6.
SEQ ID NO: 48—Safflower FAD6.
SEQ ID NO: 49—CtFAD2-2 sequence used in RNA silencing constructs.
SEQ ID NO: 50—CtFATB-3 sequence used in RNA silencing constructs.
SEQ ID NO: 51—CtFAD6 sequence used in RNA silencing constructs.
SEQ ID NO: 52—Arabidopsis thaliana oleosin promoter.
SEQ ID NO: 53-Flax lysine promoter.
SEQ ID NO: 54—Nopasynthase (Nos) polyadenylation signal.
SEQ ID NO: 55-Octopine synthase (Ocs) polyadenylation signal.
SEQ ID NO: 56- wild type CtFAD2-1 sequence corresponding to the region of the allele.
SEQ ID NO: 57—CtFAD2-1 sequence of the Ol allele with frameshift (same as S-317, CW99-OL, and LeSaf496).
SEQ ID NOs: 58-158—oligonucleotide primers.
SEQ ID NO: 159-169-Ct FAD2 enzyme motif.
SEQ ID NO: 170—wild type safflower variant SU CtFAD2-1 5′UTR intron.
SEQ ID NO: 171—High oleic safflower variant S-317 CtFAD2-1 5′UTR intron.

General Techniques and Definitions Unless otherwise specifically defined, all technical and scientific terms used herein are those of ordinary skill in the art (e.g., cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, And in biochemistry) shall be interpreted to have the same meaning as commonly understood.

  Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in the present invention are standard procedures well known to those skilled in the art. Such a technique is described, for example, in J. Org. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Am. Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T .; A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.C. M.M. Glover and B.M. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); M.M. Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current editions to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring L, Cold Spring L. E. Coligan et al. (Editors) are described and explained throughout the source literature, including Current Protocols in Immunology, John Wiley & Sons (including all current versions to date).

  The term “and / or”, for example, “X and / or Y” is taken to mean either “X and Y” or “X or Y” and is explicit to both meanings or to either meaning. Shall be construed as providing general support.

  As used herein, the term “about”, unless stated to the contrary, +/− 10% of the specified value, more preferably +/− 5%, more preferably +/− 4%, more preferably +/− 3%, more preferably +/− 2%, more preferably +/− 1.5%, more preferably +/− 1%, even more preferably +/− 0.5%.

  Throughout this specification, the term “comprise”, or variations such as “comprises” or “including,” includes elements, completeness, or steps, or elements, completeness, Alternatively, it will be understood that the inclusion of a group of steps is implied but does not imply the exclusion of any other element, integer, or step, or element, whole or group of steps.

  As used herein, the term “extracted lipid” includes at least 60% (w / w) lipid and is extracted from a transgenic organism or a portion thereof, eg, by grinding. Refers to the composition. Further, as used herein, the term “extracted oil” refers to an oil composition comprising at least 60% (w / w) oil and extracted from a transgenic organism or a portion thereof.

  As used herein, the term “purified” when used in connection with the lipids or oils of the present invention generally determines the purity of the lipid / oil component of the extracted lipids or oils. It means that it has been subjected to one or more processing steps to be enhanced. For example, the refining step can include one or more or all of the group consisting of degumming, deodorizing, decolorizing, drying, and / or fractionating the extracted oil. However, as used herein, the term “purified” refers to a transesterification process that alters the fatty acid composition of a lipid or oil of the present invention to increase the oleic acid content as a percentage of the total fatty acid content. Does not include other processes. In other words, the fatty acid composition of the refined lipid or oil is essentially the same as the fatty acid composition of the unrefined lipid or oil. The fatty acid composition, eg oleic acid, linoleic acid, and palmitic acid content of the extracted lipid or oil is essentially the same as the fatty acid composition of the lipid or oil in the plant seed from which it is obtained. In this context, “essentially the same” means +/− 1%, or preferably +/− 0.5%.

As used herein, the term “oleic acid unsaturation” or “ODP” is expressed as a percentage of the relative amounts of linoleic acid and α-linolenic acid, expressed as a percentage of the fatty acid composition of the lipid, respectively. Refers to a calculation that includes dividing by the sum of the relative amounts of oleic acid, linoleic acid, and α-linolenic acid. ceremony,
ODP = (linoleic acid (%) + α-linolenic acid (%)) / (oleic acid (%) + linoleic acid (%) + α-linolenic acid (%)).
For example, TG 603.12 (5) of Example 15 has 2.15% total linoleic acid and α-linolenic acid content, 93.88% linoleic acid, α-linolenic acid oleic acid content, and ODP 0.0229.

As used herein, the term “palmitic acid-linoleic acid-oleic acid value” or “PLO” refers to the relative amount of linoleic acid and palmitic acid expressed as a percentage of the fatty acid composition of the lipid as a percentage. Refers to a calculation that involves dividing by the relative amount of oleic acid that is made. ceremony,
PLO = (palmitic acid (%) + linoleic acid (%)) / oleic acid (%).
For example, TG603.12 (5) of Example 15 has a total linoleic and palmitic acid content of 4.71% and an oleic acid content of 91.73%, with a PLO of 0.0513.

As used herein, the term “oleic acid monounsaturation” or “OMP” refers to the relative amount of non-oleic monounsaturated fatty acids expressed as a percentage of the fatty acid composition of the lipids as a percentage. Refers to calculations involving dividing by the relative amount of oleic acid represented. ceremony,
OMP = (monounsaturated fatty acid (%)-oleic acid (%)) / oleic acid (%)
For example, TG603.12 (5) of Example 15 has a total monounsaturated fatty acid content of 91.73 excluding 1.13% oleic acid (0.84% C18: 1Δ11 + 0.29% C20: 1). % Oleic acid content with an OMP of 0.0123.

  The term “corresponding” has the same or similar genetic background as the cell of the invention, or a plant or part thereof (seed), but is not modified as described herein (eg, the invention Cells or plants or parts thereof (such as seeds) that do not have cells or plants or parts thereof that lack the exogenous polynucleotide. Corresponding cells, or plants or parts thereof (seed), for example, the amount of oleic acid produced by cells or plants or parts thereof (seed) modified as described herein, FAD2 It can be used as a control to compare one or more of activity, FATB activity, or FAD6 activity. One skilled in the art can readily determine an appropriate “corresponding” cell, plant, or part thereof (seed) for such comparison.

  As used herein, the term “seed oil” is obtained from the seed of a plant that contains at least 60% (w / w) lipid, or from the seed if the seed oil is still present in the seed. Refers to a composition that is possible. That is, seed oils consisting of or obtained using the present invention, unless specifically referred to as “extracted seed oil” or similar terminology, in seeds or parts thereof such as cotyledons or embryos In this case, it is the oil that has been extracted from the seed. The seed oil is preferably extracted seed oil. Seed oil is typically a liquid at room temperature. Preferably, the total fatty acid (TFA) content in the seed oil is greater than 70% C18 fatty acid, preferably greater than 90% oleic acid (C18: 1Δ9). The fatty acid is typically in an esterified form such as, for example, TAG, DAG, acyl-CoA, or phospholipid. Unless otherwise specified, fatty acids may be free fatty acids and / or esterified forms. In one embodiment, at least 50% of the fatty acids in the seed oil of the present invention, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99 % Can be found as TAG. In one embodiment, the seed oil of the invention is separated from one or more other lipids, nucleic acids, polypeptides, or other contaminating molecules to which it is bound in the seed or crude extract. Refined "or" refined "oil. It is preferred that the substantially refined seed oil is at least 60% free, more preferably at least 75% free, more preferably at least 90% free of other components associated therewith in the seed or extract. The seed oil of the present invention may further comprise non-fatty acid molecules, such as but not limited to sterols (see Example 17). In one embodiment, the seed oil is safflower oil (Carthamus tinctorius), sunflower oil (Helianthus annus), cottonseed oil (Gossypium hirsutum oil), Ricinus communis), rapeseed oil (Brassica napus), Brassica rapa subspecies, mustard oil (Brassica juncea), other Brassica cannabis oils (for example, Brassica cannabis) Poplarka (Brassica napobrassica), Brassica camelina (Brassica camelina)), Amama Oil (Linum usitatissimum), soybean oil (Glycine max), corn oil (Zea mays), tobacco oil (Nicotiana tabacum), peanut oil (Arakishypogea) Arachis hypogaea), coconut oil (Elaeis guineensis), coconut oil (Cocos nucifera), avocado oil (Persea americana) Europa oil, olea oil europaea)), cashew oil (Anacardium occidentale) ), Macadamia oil (Macadamia interglifolia), almond oil (Prunus amygdalus), oat seed oil (Avena sativa), rice a Or Oriza graberima), camelina oil (Camelina sativa), hamana oil (Crambe abyssinica), or Arabidopsis apsia seed oil (Arabidopsis apsid) is there. Seed oil can be extracted from the seed by any method known in the art. This generally involves extraction with a non-polar solvent such as hexane, diethyl ether, petroleum ether, chloroform / methanol or butanol mixtures, typically in connection with the initial grinding or rolling of the seed. Lipids associated with starch in cereals can be extracted with water-saturated butanol. Seed oil can be “degummed” by methods known in the art to remove polysaccharides and / or phospholipids, or it can be free of contaminants, purity, stability, or It can be processed in other ways to improve color. TAG and other esters in seed oil can be hydrolyzed to release free fatty acids, for example, by acid or alkali treatment, or by lipase action, or seed oil can be hydrogenated, or It can be treated chemically or enzymatically as is known in the art. However, once the seed oil has been treated, it no longer contains TAG and is no longer considered a seed oil as referred to herein.

  Free and esterified sterols in purified and / or extracted lipids or oils (eg, sitosterol, campesterol, stigmasterol, brassicasterol, Δ5-avenasterol, sitostanol, campestanol, and cholesterol) Concentrations were measured in Phillips et al. (2002) and / or provided in Example 17. Sterols in vegetable oils exist as free alcohols, fatty acid esters (esterified sterols), sterol glycosides and acylated glycosides. The sterol concentration in natural vegetable oil (seed oil) ranges up to about 1100 mg / 100 g. Hydrogenated coconut oil has one of the lowest natural vegetable oil concentrations at about 60 mg / 100 g. The recovered or extracted seed oil of the present invention preferably has from about 100 to about 1000 mg total sterol / 100 g (oil). For food or diet, sterols exist primarily as free or esterified forms rather than glycosylated forms. In the seed oil of the present invention, preferably at least 50% of the sterols in the oil are present as esterified sterols, except for soybean seed oil which has about 25% of the esterified sterols. The safflower seed oil of the present invention preferably has from about 150 to about 400 mg total sterol / 100 g, typically about 300 mg total sterol / 100 g seed oil, with the main sterol having sitosterol. Rapeseed seed oil and rapeseed oil of the present invention preferably have from about 500 to about 800 mg total sterol / 100 g, with the main sterol having sitosterol and then the most abundant campesterol. The corn seed oil of the present invention preferably has from about 600 to about 800 mg total sterol / 100 g, with the main sterol having sitosterol. The soybean seed oil of the present invention preferably has about 150 to about 350 mg of total sterol / 100 g, with the main sterol having sitosterol and then the most abundant stigmasterol, more than the esterified sterol. It also has free sterols. The cottonseed oil of the present invention preferably has from about 200 to about 350 mg total sterol / 100 g, with the main sterol having sitosterol. The coconut oil and coconut oil of the present invention preferably has from about 50 to about 100 mg total sterol / 100 g, with the main sterol having sitosterol. The peanut seed oil of the present invention preferably has from about 100 to about 200 mg total sterol / 100 g, with the main sterol having sitosterol. The sesame seed oil of the present invention preferably has from about 400 to about 600 mg total sterol / 100 g, with the main sterol having sitosterol. The sunflower seed oil of the present invention preferably has from about 200 to about 400 mg total sterol / 100 g, with the main sterol having sitosterol.

  As used herein, the term “fatty acid” refers to a carboxylic acid having a long aliphatic terminus consisting of at least 8 carbon atoms in length, either saturated or unsaturated. Typically, fatty acids have a carbon-carbon linked chain of at least 12 carbons in length. Most natural fatty acids have an even number of carbon atoms because their biosynthesis includes acetates with two carbon atoms. The fatty acids can be in the free (non-esterified) or esterified form, eg, TAG, DAG, MAG, part of an acyl-CoA (thioester) bond, or other covalent form. When covalently linked in esterified form, the fatty acid is referred to herein as an “acyl” group. Fatty acids can be esterified as phospholipids such as, for example, phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidyllinositol, or diphosphatidylglycerol. Saturated fatty acids do not contain any double bonds or other functional groups along the chain. The term “saturated” refers to hydrogen in that every carbon (apart from the carboxylic acid [—COOH] group) contains as much hydrogen as possible. That is, the omega (ω) end contains 3 hydrogens (CH 3 —) and each carbon in the chain contains 2 hydrogens (—CH 2 —). Unsaturated fatty acids have one or more alkene functional groups present along the chain, and each alkene replaces a single bond “—CH 2 —CH 2 —” portion of the chain with a double bond “—CH═CH—” portion (ie, It is a form similar to a saturated fatty acid except that it is substituted with a carbon that is double-bonded to another carbon). Two adjacent carbon atoms in the chain attached to either side of the double bond can appear in a cis or trans configuration.

  As used herein, the term “polyunsaturated fatty acid” or “PUFA” refers to at least 12 carbon atoms and at least 2 alkene groups (carbon-carbon double bonds) in the carbon chain. Refers to fatty acids containing.

  As used herein, the term “monounsaturated fatty acid” refers to a single double bond in their acyl chains such as oleic acid (C18: 1Δ9), C18: 1D11, and C20: 1. The fatty acid which has.

  “Triacylglycerides” or “TAGs” are glycerides in which glycerol is esterified with three fatty acids. In the Kennedy pathway of TAG synthesis, DAG is formed as described above, and then a third acyl group is esterified to the glycerol backbone by the activity of DGAT. Alternative pathways for the formation of TAGs include those catalyzed by the enzymes PDAT and MGAT pathway (PCT / AU2011 / 000794).

  “Diacylglycerides” or “DAGs” are glycerides in which glycerol is esterified with two fatty acids. As used herein, DAG contains a hydroxyl group at the sn-1,3 or sn-1,2,2,3 positions, and therefore DAG does not contain phosphorylated molecules such as PA or PC. Thus, DAG is a component of neutral lipids in cells. In the Kennedy pathway of DAG synthesis, the precursor sn-glycerol-3-phosphate (G-3-P) is esterified to two acyl groups, each derived from a fatty acid coenzyme A ester, in the first reaction A second catalyzed by glycerol-3-phosphate acyltransferase (GPAT) at the sn-1 position to form LysoPA and subsequently catalyzed by lysophosphatidic acid acyltransferase (LPAAT) at the sn-2 position. Acylation forms phosphatidic acid (PA). This intermediate is then dephosphorylated to form DAG. In an alternative assimilation pathway, DAG can be formed by acylation of either sn-1 MAG, or preferably sn-2 MAG, catalyzed by MGAT. DAG can also be formed from TAG by removal of the acyl group by lipase, or essentially from PC by removal of the choline head group by any of the enzymes CPT, PDCT, or PLC.

  As used herein, the term “desaturase” is typically an enzyme that can introduce a carbon-carbon double bond into an acyl group of a fatty acid substrate, for example, an esterified form such as a fatty acid CoA ester. Point to. Acyl groups can be esterified to phospholipids such as phosphatidylcholine (PC), to acyl carrier protein (ACP), to CoA, or in preferred embodiments to PC. Thus, desaturases can generally be classified into three groups. In one embodiment, the desaturase is a front end desaturase.

The term "Δ12 desaturase" and "FAD2" as used herein, oleic acid ^(18: 1 Δ9) linoleic acid ^(C18: 2 Δ9,12) film performs desaturase reaction to convert the binding Δ12 fatty Desuchuraze (Desturase). Thus, the term “Δ12 desaturase activity” refers to the conversion of oleic acid to linoleic acid. These fatty acids can be in esterified form, for example as part of a phospholipid, preferably in the form of PC. In one embodiment, the FAD2 enzyme as defined herein comprises three histidine rich motifs (His boxes) (see Table 5 for examples of His boxes of enzymes of the invention). Such His-rich motifs are highly conserved in the FAD2 enzyme and are involved in the formation of ferric oxide complexes used in biochemical catalysis (Shanklin et al., 1998).

  As used herein, the terms “FAD2-1” and “CtFAD2-1” and variations thereof are provided as safflower FAD2 polypeptide, for example, SEQ ID NO: 12, with the amino acid sequence provided as SEQ ID NO: 27. Refers to a polypeptide encoded by a nucleotide having a sequence. As used herein, a FAD2-1 gene is a gene that encodes an allele due to such a polypeptide or a mutation thereof. These terms also include variants that are naturally or artificially derived or generated from sequences provided. In one embodiment, the FAD2-1 of the invention comprises an amino acid sequence that is at least 95% identical, more preferably at least 99% identical to the sequence provided as SEQ ID NO: 27. The CtFAD2-1 gene includes an allele by a mutant encoding a polypeptide having an alteration in desaturase activity such as a decrease in activity, or an allele that does not encode a functional polypeptide (null allele). Such alleles can be natural or induced by artificial mutagenesis. An example of such an allele is the FAD2-1ol allele described herein.

  As used herein, the terms “FAD2-2” and “CtFAD2-2”, and variations thereof, are provided as safflower FAD2 polypeptide, for example, SEQ ID NO: 13, with the amino acid sequence provided as SEQ ID NO: 28. Refers to a polypeptide encoded by a nucleotide having a sequence. As used herein, a FAD2-2 gene is a gene that encodes an allele due to such a polypeptide or a mutation thereof. These terms also include variants that are naturally or artificially derived or generated from sequences provided. In one embodiment, the FAD2-2 of the invention comprises an amino acid sequence that is at least 95% identical, more preferably at least 99% identical to the sequence provided as SEQ ID NO: 28. CtFAD2-2 genes include alleles with mutants encoding polypeptides having altered desaturase activity alterations, such as decreased activity, or alleles that do not encode functional polypeptides (null alleles) . Such alleles can be natural or induced by artificial mutagenesis.

  The terms “FAD2-10” and “CtFAD2-10” as used herein, and variations thereof, are provided as safflower FAD2 polypeptide, for example, SEQ ID NO: 21, whose amino acid sequence is provided as SEQ ID NO: 36. Refers to a polypeptide encoded by a nucleotide having a sequence. As used herein, a FAD2-10 gene is a gene that encodes an allele due to such a polypeptide or a mutation thereof. These terms also include variants that are naturally or artificially derived or generated from sequences provided. In one embodiment, the FAD2-10 of the invention comprises an amino acid sequence that is at least 95% identical, more preferably at least 99% identical to the sequence provided as SEQ ID NO: 36. The CtFAD2-10 gene includes an allele by a mutant encoding a polypeptide having an alteration in desaturase activity such as a decrease in activity, or an allele that does not encode a functional polypeptide (null allele). Such alleles can be natural or induced by artificial mutagenesis.

  As used herein, the terms “palmitoyl-ACP thioesterase” and “FATB” refer to proteins that hydrolyze palmitoyl-ACP to produce free palmitic acid. Thus, the term “palmitoyl-ACP thioesterase activity” refers to the hydrolysis of palmitoyl-ACP to produce free palmitic acid.

  As used herein, the terms “FATB-3” and “CtFATB-3” and variations thereof are provided as safflower FATB polypeptide, eg, SEQ ID NO: 43, whose amino acid sequence is provided as SEQ ID NO: 45. Refers to a polypeptide encoded by a nucleotide having a sequence. As used herein, a FATB-3 gene is a gene that encodes an allele due to such a polypeptide or a mutation thereof. These terms also include variants that are naturally or artificially derived or generated from sequences provided. In one embodiment, the FATB-3 of the invention comprises an amino acid sequence that is at least 95% identical, more preferably at least 99% identical to the sequence provided as SEQ ID NO: 45. The CtFATB-3 gene includes an allele due to a mutant encoding a polypeptide having a modified palmitoyl-ACP thioesterase activity, such as decreased activity, or an allele that does not encode a functional polypeptide (null allele) included. Such alleles can be natural or induced by artificial mutagenesis.

  As used herein, “plastid ω6 fatty acid desaturase”, variations thereof, and “FAD6” include all phosphatidylglycerols, monogalactosyldiacylglycerols, digalactosyldiacylglycerols, and sulfoguinovosyl diacylglycerols, respectively. Refers to chloroplast enzymes that desaturate 16: 1 and 18: 1 fatty acids to 16: 2 and 18: 2 in chloroplast membrane lipids containing 16: 1 or 18: 1.

  As used herein, the terms “FAD6” and “CtFAD6” and variations thereof include a safflower FAD6 polypeptide whose amino acid sequence is provided as SEQ ID NO: 48, eg, a nucleotide having the sequence provided as SEQ ID NO: 47. Refers to the polypeptide encoded by. As used herein, a FAD6 gene is a gene that encodes an allele due to such a polypeptide or a mutation thereof. These terms also include variants that are naturally or artificially derived or generated from sequences provided. In one embodiment, the FAD6 of the invention comprises an amino acid sequence that is at least 95% identical, more preferably at least 99% identical to the sequence provided as SEQ ID NO: 48. The CtFAD6 gene includes an allele by a mutant encoding a polypeptide having a modification of desaturase activity such as a decrease in activity, or an allele that does not encode a functional polypeptide (null allele). Such alleles can be natural or induced by artificial mutagenesis.

  As used herein, the term “acetylenase” or “fatty acid acetylenase” refers to an enzyme that introduces a triple bond into a fatty acid, resulting in the production of an acetylenic fatty acid.

  The term “Ct” is used herein before terms such as FAD2, FATB, and FAD6 to indicate that the enzyme / gene is from safflower.

  As used herein, the term “silencing RNA capable of reducing the expression of” and variations thereof refer to endogenous enzymes such as Δ12 desaturase, palmitoyl-ACP thioesterase, plastid ω6 fatty acid desaturase, or these Decrease (down-regulate) production and / or activity (e.g., encode siRNA, hpRNAi), or as such, production and / or activity (e.g., combination of two or more or all three) A polynucleotide encoding an RNA molecule that down-regulates (eg, siRNA) that can be delivered directly to a cell. In one embodiment, the silencing RNA is generated from a transgene in the cell, eg, endogenously generated in the cell by reducing the amount of mRNA encoding the endogenous enzyme or reducing its translation. An exogenous RNA that reduces the amount of sex enzyme at the transcriptional stage and / or after transcription. Silencing RNA is typically 21-24 nucleotides in length that is complementary to endogenous mRNA and can be associated with a silencing complex known as RISC in the cell.

As used herein, the term “mutant (s) that reduces activity” refers to the amino acid sequence provided phenotypically as normal seeds (eg, SEQ ID NOs: 27, 28, and 36). Natural or artificial mutants having a lower level of defined enzyme activity (eg FAD2 enzyme activity in seeds) (eg chemicals) Or by site-directed mutagenesis). Examples of phenotypically common safflower subspecies include, but are not limited to, Centennial, Finch, Nutrasaff, and Cardinal. The first identified high oleic acid characteristics in safflower found in safflower introduction from India were dominated by a partially recessive allele designated ol at a single locus OL (Knowles and Hill) , 1964). The OL locus described herein corresponds to the CtFAD2-1 gene. The oleic acid content of seed oil in the olol genotype was typically 71-75% in greenhouse-grown plants (Knowles, 1989). Knowles (1968) incorporated the ol allele into the safflower breeding program and in the United States released the first high oleic acid (HO) safflower variant “UC-1” in 1966, followed by improved variants Saffola series including “Oleic Leed” and Saffola 317 (S-317), S-517, and S-518 were released. Their high oleic acid (olol) genotype was relatively stable at the oleic acid level when grown at different temperatures in the field (Bartholomew, 1971). In addition, Knowles (1972) also described different alleles ol ₁ at the same locus generated with 35-50% oleic acid homozygous conditions. In contrast to olol genotype, ol ₁ ol ₁ genotype showed a strong response to temperature (Knowles, 1972). In the safflower seeds as determined herein, the allele of the ol mutant that results in decreased FAD2-1 activity (and total FAD2 activity) is a frameshift mutation (single single A mutant FAD2-1 gene containing (by nucleotide deletion) (see also Example 7 and SEQ ID NOs: 26 and 38).

  As used herein, the expression "can produce a plant that produces seeds that the oil content contains" or "can produce a plant that produces oil seeds that contain oil" or variations thereof Are defined components when a plant produced from seed, preferably an oilseed plant, more preferably a safflower plant, is grown under optimal conditions, e.g., greenhouse conditions such as those referred to in the examples. It has the ability to produce oil using. If you own seeds from plants, use standard procedures such as those described herein to develop progeny plants from at least one of the seeds under suitable greenhouse conditions; It is normal to test for oil content and fatty acid composition in seed oil from progeny plants. Thus, as those skilled in the art will appreciate, seeds grown outdoors are defined herein by undesired conditions in a particular year such as heat waves, cold waves, droughts, floods, frosts, pest stress, etc. May not meet all the required requirements, but if the seed is grown under more desirable conditions, it can produce progeny plants that produce the defined oil-containing and fatty acid composition, Such seeds are nevertheless encompassed by the present invention.

  As used herein, the term “weight” refers to a substance (eg, oleic acid, palmitic acid, or PUFA, eg, linoleic acid or linolenic acid, as a percentage of the weight of the composition, including the ingredients of the substance or composition. Acid). For example, the weight of a particular fatty acid such as oleic acid can be measured as the total fatty acid content of the lipid or seed oil, or as a percentage of the weight of the seed.

  As used herein, the term “biofuel” is typically used in power machines such as automobiles, trucks, or oil-powered motors, whose energy is derived from fossil fuels. Rather, it refers to any kind of fuel that is derived from biological carbon fixation. Biofuels include fuels derived from biomass conversion, as well as solid biomass, liquid fuels, and biogas. Examples of biofuels include bioalcohol, biodiesel, synthetic diesel, vegetable oil, bioether, biogas, synthesis gas, solid biofuel, algae-derived fuel, biohydrogen, biomethanol, 2,5-dimethylfuran (DMF) ), Biodimethyl ether (bioDME), Fischer-Tropsch diesel, biohydrogen diesel, mixed alcohols, and wood diesel.

  As used herein, the term “industrial product” refers to, for example, fatty acid methyl- and / or ethyl-esters, or alkanes such as methane, mixtures of long-chain alkanes that are normally liquid at ambient temperature, biofuels, Refers to hydrocarbon products made primarily from carbon and hydrogen, such as carbon monoxide and / or hydrogen, or bioalcohols such as ethanol, propanol, or butanol, or biochar. The term “industrial product” is intended to include intermediate products that can be converted to other industrial products, for example, synthesis gas itself is a hydrocarbon product (also an industrial product). Is considered an industrial product that can be used to synthesize. As used herein, the term industrial product includes both pure forms of the above compounds or, more generally, mixtures of various compounds and ingredients, as understood in the art. As such, for example, the hydrocarbon product may include a range of carbon chain lengths.

The term polynucleotide "polynucleotide" and "nucleic acid" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. The polynucleotides of the present invention, depending on their origin or manipulation, may be (1) not related to all or part of a naturally related polynucleotide or (2) linked to a polynucleotide other than that naturally linked Or (3) non-naturally occurring, genomic, cDNA, semi-synthetic, or synthetic origin, double-stranded or single-stranded. The following are non-limiting examples of polynucleotides: the coding or non-coding region of a gene or gene fragment, the locus (s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA ( tRNA), ribosomal RNA (rRNA), ribozyme, cDNA, recombinant polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, chimeric DNA of any sequence, nucleic acid probe, and primer. Preferred polynucleotides of the invention include double stranded DNA molecules that can be transcribed in plant cells, and silencing RNA molecules.

  As used herein, the term “gene” is to be construed in its broadest context, including the transcription region of a structural gene and, if translated, the protein coding region, at least about at any end. It contains a deoxyribonucleotide sequence that is located adjacent to the coding region on both 5 ′ and 3 ′ ends over a 2 kb distance and that contains sequences involved in gene expression. In this regard, a gene includes a control signal such as a promoter, enhancer, termination, and / or polyadenylation signal that is naturally associated with a given gene, or includes a heterologous control signal, in which case the gene is a “chimeric” Referred to as "gene". Sequences located 5 'of the protein coding region and present on the mRNA are referred to as 5' untranslated sequences. Sequences located 3 'or downstream of the protein coding region and present on the mRNA are referred to as 3' untranslated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene includes a coding region that can be interrupted by non-coding sequences termed “introns”, “intervening regions”, or “intervening sequences”. Introns are segments of genes that are transcribed into nuclear RNA (nRNA). Introns can contain regulatory elements such as enhancers. Introns are removed or “spliced” from the nucleus or from the primary transcript, so that introns are not present in the mRNA transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term “gene” includes synthetic or fusion molecules that encode all or part of the proteins of the invention described herein and nucleotide sequences that are complementary to any one of the above.

  “Allele” refers to one specific form (eg, gene) of a gene sequence within a cell, individual plant, or population, wherein the specific form includes at least one, often one, within the sequence of the gene. It differs from other forms of the same gene in the sequence of the mutation site that exceeds. The sequences of these mutation sites that differ between different alleles are referred to as “variances”, “polymorphisms”, or “mutations”.

  As used herein, “chimeric DNA” refers to virtually any DNA molecule not found in nature and is referred to herein as “DNA constructs”. Typically, chimeric DNA contains regulatory and transcriptional or protein coding sequences that are not actually found together. Thus, chimeric DNA can include regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source, but arranged in a manner different from that actually found. An open reading frame may or may not be linked to its natural upstream and downstream regulatory elements. An open reading frame can be incorporated, for example, in a plant genome where it is not found in nature, or in a replicon or vector where it is not found in nature, such as a bacterial plasmid or viral vector. The term “chimeric DNA” is not limited to DNA molecules that are capable of replicating in a host, but includes, for example, DNA that can be ligated into a replicon by a specific adapter sequence.

  A “transgene” is a gene that has been introduced into the genome by a transformation procedure. The transgene may be an early transformed plant produced by regeneration from transformed plant cells, or a progeny plant produced by self-fertilization or mating from an early transformed cell, or a plant part such as a seed. It can be inside. The term “genetically modified” and variations thereof refer to introducing a gene into a cell by transformation or transduction, mutating a gene in a cell, and a gene in a cell or as described above. Genetic modification or regulation of the gene regulation of any modified cell progeny.

  As used herein, a “genomic region” is a transgene or group of transgenes (also referred to herein as a cluster) that is inserted into a cell or its ancestor so that they are post-meiotic. , Refers to a position in the genome that is inherited together in progeny cells.

  A “recombinant polynucleotide” or “exogenous polynucleotide” of the present invention refers to a nucleic acid molecule constructed or modified by artificial recombination methods. A recombinant polynucleotide can be present in a cell in a modified amount or expressed at a modified rate (eg, in the case of mRNA) compared to its native state. An exogenous polynucleotide is a polynucleotide that is introduced into a cell that does not naturally contain the polynucleotide. Typically, exogenous DNA is used as a template for transcription of mRNA and is translated into a contiguous sequence of amino acid residues encoding the polypeptide of the invention in the transformed cell. In another embodiment, a portion of the exogenous polynucleotide is endogenous to the cell and its expression is altered by recombinant means, eg, an exogenous regulatory sequence is introduced upstream of the endogenous polynucleotide. To allow the transformed cells to express the polypeptide encoded by the polynucleotide. For example, an exogenous polynucleotide can cause antisense RNA to be expressed in an endogenous polynucleotide.

  Recombinant polynucleotides of the invention are produced in a cell-based or cell-free expression system that is not separated from other components of the cell-based or cell-free expression system in which the polynucleotide is present, and thereafter at least Polynucleotides purified from several other components. A polynucleotide can be a stretch of contiguous nucleotides that occur in nature, or two or more contiguous sources from different sources (natural and / or synthetic) joined together to form a single polynucleotide. It can contain a stretch of nucleotides. Typically, such a chimeric polynucleotide is at least one encoding a polypeptide of the invention operably linked to a promoter suitable for activating transcription of an open reading frame in the cell of interest. Includes two open reading frames.

  It will be appreciated that for a defined polynucleotide, a higher% identity number than those provided above encompasses preferred embodiments. Thus, where applicable, considering the minimum% identity number, a polynucleotide is at least 50%, more preferably at least 60%, more preferably at least 65%, more preferably At least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 9 0.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more It is preferred to include polynucleotide sequences that are preferably at least 99.7% identical, more preferably at least 99.8%, and even more preferably at least 99.9% identical.

  A polynucleotide of or useful for the present invention can be selectively hybridized to a polynucleotide as defined herein under stringent conditions. As used herein, stringent conditions include: (1) denaturing agents such as formamide during hybridization, eg, 0.1% (w / v) bovine serum albumin at 42 ° C. Using 50% (v / v) formamide containing 1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 containing 750 mM NaCl, 75 mM sodium citrate, Or (2) 50% formamide, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM phosphoric acid at 42 ° C. in 0.2 × SSC and 0.1% SDS Sodium (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon sperm DNA (50 g / ml), % SDS, and 10% dextran sulfate and / or (3) 0.015 M NaCl / 0.0015 M sodium citrate / low ionic strength and high temperature for washing, eg 50 ° C. Use 0.1% SDS.

A polynucleotide of the present invention may have one or more mutations that are deletions, insertions, or substitutions of nucleotide residues when compared to the natural molecule. A polynucleotide having a mutation relative to a reference sequence can be naturally occurring (ie, isolated from a natural source) or (eg, as described above, site-directed mutagenesis on a nucleic acid or It can be synthetic (by performing DNA shuffling).

Polynucleotides for reducing the expression level of endogenous proteins In one embodiment, the cells / seed / plants / organisms of the invention comprise an introduced mutant or exogenous polynucleotide to produce endogenous enzymes and Increased production of oleic acid, preferably palmitic acid and PUFA, such as when compared to corresponding cells that down-regulate activity, typically lacking introduced mutants or exogenous polynucleotides This leads to a decrease in the production of linoleic acid. Examples of such polynucleotides include antisense polynucleotides, sense polynucleotides, catalytic polynucleotides, microRNAs, polynucleotides encoding polypeptides that bind to endogenous enzymes, and double stranded RNA.

RNA interference RNA interference (RNAi) is particularly useful for specifically inhibiting the production of certain proteins. This technique relies on the presence of a dsRNA molecule comprising a sequence that is essentially identical to and complementary to the mRNA of the gene of interest or part thereof. Conveniently, dsRNA can be generated from a single promoter in a recombinant vector or host cell, and the sense and antisense sequences are covalently linked by a sequence, preferably an unrelated sequence, and the target RNA or its complement. A sequence having identity to can hybridize to form a dsRNA molecule having a binding sequence in which the sense and antisense sequences in the corresponding transcript form a loop structure. to enable. Typically, a dsRNA is encoded by a double stranded DNA construct having sense and antisense sequences in an inverted repeat structure arranged as an interrupted palindrome, the repeat sequence hybridizing within the dsRNA molecule. The interrupting sequence is transcribed to form a loop in the dsRNA molecule. The design and generation of suitable dsRNA molecules is well within the ability of those skilled in the art, specifically, Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619, WO 99/53050, WO 99/49029, and WO 01/34815.

  In one example, DNA that induces the synthesis of at least 19 contiguous nucleotides that are complementary to a region of the target RNA to be inactivated, preferably at least partially double-stranded RNA product (s) that have homology. Is introduced. Thus, when DNA is transcribed into RNA, it contains both sense and antisense sequences that hybridize to form double stranded RNA regions. In one embodiment of the invention, the sense and antisense sequences are separated by a spacer region that contains the joined intron when transcribed into RNA. This arrangement has been shown to result in higher efficiency gene silencing. A double-stranded RNA region can include one or two RNA molecules transcribed from one or two DNA regions. The presence of a double-stranded molecule is thought to cause a response from an endogenous system that destroys both double-stranded RNA and homologous RNA transcripts from the target gene, effectively reducing or losing the activity of the target gene. It is done.

  The lengths of the hybridizing sense and antisense sequences must each be at least 19 contiguous nucleotides corresponding to a portion of the target mRNA. Full length sequences corresponding to the entire gene transcript can be used. The degree of identity of the sense and antisense sequences to the target transcript must be at least 85%, at least 90%, or at least 95% to 100%. Of course, an RNA molecule can contain unrelated sequences that can act to stabilize the molecule. The RNA molecule can be expressed under the control of an RNA polymerase II or RNA polymerase III promoter. Examples of the latter include tRNA or snRNA promoters.

  Preferred small interfering RNA (“siRNA”) molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA. Preferably, the siRNA sequence begins with dinucleotide AA and includes a GC content of about 30-70% (preferably 30-60%, more preferably 40-60%, more preferably about 45% -55%). For example, it does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the organism into which it is introduced, as measured by a standard BLAST search.

  As an example, a dsRNA of the invention comprises a nucleotide sequence provided as any one of SEQ ID NOs: 49-51 (each T is U).

MicroRNA
MicroRNAs (abbreviated miRNAs) are generally non-coding RNA molecules derived from larger precursors that form an incomplete stem-loop structure of 19-25 nucleotides (usually 20-24 nucleotides in plants).

  miRNAs bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing.

  In plant cells, miRNA precursor molecules are thought to be processed first in the nucleus. A pri-miRNA (which includes one or more locally double-stranded or “hairpin” regions and usually a 5 ′ “cap” and a polyadenylated tail of the mRNA) also includes a stem loop or folded structure, Processed into shorter miRNAs referred to as “pre-miRNAs”. In plants, pre-miRNAs are cleaved by different DICER-like (DCL) enzymes, specifically DCL-1, to obtain miRNA: miRNA * duplexes. Prior to nuclear export, these duplexes are methylated. In contrast, hairpin RNA molecules with longer dsRNA regions are specifically processed by DCL-3 and DCL-4.

  In the cytoplasm, miRNA strands from miRNA: miRNA duplexes are selectively incorporated into active RNA-induced silencing complexes (RISC) for target recognition. RISC complexes contain a specific subset of Argonaute proteins that exhibit sequence-specific gene repression after transcription (see, eg, Millar and Waterhouse, 2005, Pasquinelli et al., 2005, Almeida and Allshire, 2005). See).

Co-suppression genes can suppress the expression of related endogenous genes and / or transgenes already present in the genome, a phenomenon called homology-dependent gene silencing. Most examples of homology-dependent gene silencing fall into two classes: those that function at the transgene transcription level and those that are manipulated after transcription.

  Post-transcription homology-dependent gene silencing (ie, co-suppression) explains the loss of expression of the transgene and related endogenous or viral genes in transgenic plants. Co-suppression often occurs when the transcript of the transgene is abundant, but not necessarily, as it is generally caused at the level of mRNA processing, localization, and / or degradation. Conceivable. The Severa1 model exists to explain how co-suppression works (see Taylor, 1997).

  Co-suppression involves introducing an extra copy of a gene or fragment thereof into a plant in sense orientation relative to a promoter for its expression. One skilled in the art will appreciate that the size of the sense fragment, its degree of correspondence to the target gene region, and the degree of sequence identity with the target gene can vary. In some cases, the additional copy of the gene sequence interferes with the expression of the target plant gene. Reference is made to WO 97/20936 and EP 0465572 for methods of implementing a co-suppression approach.

Expression Vector As used herein, an “expression vector” is a DNA or RNA vector that can transform a host cell and effect the expression of one or more specific polynucleotides. Preferably, the expression vector can replicate in the host cell or be integrated into the host cell genome. The expression vector is typically a virus or a plasmid. Expression vectors of the present invention include any vector that functions (ie, induces gene expression) in the host cells of the present invention, including fungi, algae, and plant cells.

  As used herein, “operably linked” refers to a functional relationship between two or more nucleic acid (eg, DNA) segments. Typically, it refers to the functional relationship of a transcription regulatory element (promoter) to a transcription sequence. For example, a promoter is operably linked to a polynucleotide coding sequence as defined herein if it stimulates or regulates transcription of the coding sequence in a suitable cell. In general, promoter transcription regulatory elements operably linked to a transcription sequence are physically adjacent to the transcription sequence, ie, they are cis-acting. However, some transcription regulatory elements, such as enhancers, do not need to be physically adjacent to or in close proximity to coding sequences that enhance transcription.

  The expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and control the expression of the polynucleotides of the invention. In particular, the expression vector of the present invention includes a transcription control sequence. A transcription control sequence is a sequence that controls the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those that control transcription initiation such as promoter, enhancer, operator, and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the invention. The choice of regulatory sequences used depends on the target organism, such as the plant of interest and / or the target organ or tissue. Such regulatory sequences can be obtained from any eukaryotic organism such as a plant or plant virus, or can be chemically synthesized. A variety of such transcription control sequences are known to those skilled in the art. Particularly preferred transcription control sequences are promoters that are active in inducing transcription in plants, constitutively or stage and / or tissue-specific, depending on the use of the plant or part (s) thereof. .

  Many vectors suitable for stable transfection of plant cells or for the construction of transgenic plants are described, for example, in Pouwels et al. , Cloning Vectors: A Laboratory Manual, 1985, sup. 1987, Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989, and Gelvin et al. , Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under transcriptional control of 5 'and 3' regulatory sequences, and a dominant selectable marker. Such plant expression vectors include promoter regulatory regions (eg, regulatory regions that control inducible or constitutive, environmental or developmental regulation, or cell or tissue specific expression), transcription initiation sites, ribosome binding sites, RNA Processing signals, transcription termination sites, and / or polyadenylation signals can also be included.

  A number of constitutive promoters that are active in plant cells have been described. Examples of promoters suitable for constitutive expression in plants include cauliflower mosaic virus (CaMV) 35S promoter, cynomolgus mosaic virus (FMV) 35S, sugarcane rod-shaped virus promoter, communis yellow spotted virus promoter, ribulose-1,5-bis-phosphorus Light inducible promoter from small subunit of acid carboxylase, rice cytosol triose phosphate isomerase promoter, Arabidopsis adenine phosphoribosyltransferase promoter, rice actin 1 gene promoter, mannopine synthase and octopine synthase promoter, Adh promoter, sucrose Synthase promoter, R gene complex promoter, and chlorophyll α / β binding protein inheritance Including, but not limited to, child promoters. These promoters have been used to create DNA vectors that are expressed in plants, see, eg, International Publication No. WO 84/02913. All of these promoters have been used to create various types of plant-expressing recombinant DNA vectors.

  For the purposes of expression of plant source tissues, such as leaves, seeds, roots or stems, the promoter utilized in the present invention preferably has a relatively high expression in these specific tissues. For this purpose, one can choose from a number of promoters for genes specific for tissue or cells or with enhanced expression. Examples of such promoters reported in the literature include pea-derived chloroplast glutamine synthetic GS2 promoter, wheat-derived chloroplast fructose-1,6-biphosphatase promoter, potato-derived nuclear photosynthetic ST-LS1 promoter , The serine / threonine kinase promoter, and the glucoamylase (CHS) promoter from Arabidopsis thaliana.

  (1) heat, (2) light (eg, pea RbcS-3A promoter, corn RbcS promoter), (3) hormones such as abscisic acid, (4) damage (eg, WunI), or (5) chemicals, eg Various plants that are regulated in response to environmental, hormonal, chemical, and / or developmental signals, including promoters regulated by methyl jasmonate, salicylic acid, steroid hormones, alcohol, Safeners (WO 97/06269) Gene promoters can also be used for expression of RNA binding protein genes in plant cells, or (6) it may be advantageous to use organ specific promoters.

  The term “plant storage organ-specific promoter” as used herein refers to a promoter that selectively induces gene transcription in a plant storage organ when compared to other plant tissues. The plant storage organ is preferably a seed. Preferably, the promoter only induces the expression of the gene of interest in the storage organ, and / or the expression of the gene of interest in other parts of the plant, for example leaves, is subject to Northern blot analysis and / or RT -Undetectable by PCR. Typically, a promoter activates gene expression during storage organ growth and development, particularly during the synthesis and accumulation phase of storage compounds in the storage organ. Such promoters are gene expression in the whole plant storage organ or only part thereof, for example, seed coat, embryo, or cotyledon (s) in dicotyledonous seeds, or endosperm or aleurone layer of monocotyledonous seeds Can be active.

  Other promoters can also be used to express the protein in specific tissues, such as seeds or fruits. Promoters for β-conglycinin or other seed-specific promoters such as napin, zein, linin, and phaseolin promoters can be used. In one embodiment, the promoter induces expression in tissues and organs where lipid biosynthesis occurs. Such promoters act in seed development at the appropriate time to modify the lipid composition in the seed. In one embodiment, the plant storage organ specific promoter is a seed specific promoter. In a more preferred embodiment, the promoter selectively induces the expression of dicotyledonous embryos and / or cotyledons, or monocotyledonous endosperm, compared to other organs in the plant, such as leaves. Preferred promoters for seed-specific expression include 1) promoters from genes encoding enzymes involved in lipid biosynthesis and accumulation in seeds such as desaturases and elongases, 2) from genes encoding seed storage proteins Promoters, and 3) promoters from genes encoding enzymes involved in carbohydrate biosynthesis and accumulation in seeds. Seed-specific promoters that are suitable are flax linin promoters (eg Cnl1 or Cnl2) rapeseed napin gene promoter (US Pat. No. 5,608,152), Vicia faba USP promoter (Baumlein et al., 1991). The Arabidopsis oleosin promoter (International Publication No. WO 98/45461), the Phaseolus vulgaris phaseolin promoter (US Pat. No. 5,504,200), the Brassica Bce4 promoter ( WO91 / 13980), or legumin B4 promoter (Baumlein et al., 1992), as well as maize and barley Is a promoter that provides wheat, rye, seed-specific expression in monocots such as rice. Prominent promoters that are suitable are the barley lpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230) or the promoters described in WO 99/16890 (barley hordein gene, Rice glutelin gene, rice lysine gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, corn zein gene, oats glutelin gene, sorghum saccharin gene, rye secarin gene-derived promoter). Other promoters include Broun et al. (1998), Potenza et al. (2004), U.S. Patent No. 20070192902, and U.S. Patent No. 20030159173. In one embodiment, the seed specific promoter is selectively expressed in a defined portion of the seed, such as an embryo, cotyledon (s), or endosperm. Cotyledon-specific promoters include the FP1 promoter (Ellerstrom et al., 1996), the pea legumin promoter (Perrin et al., 2000), and the kidney bean phytohemagglutinin promoter (Perrin et al., 2000). Including, but not limited to. In further embodiments, the seed-specific promoter is not expressed or only expressed at low levels in the embryo and / or after the seed has germinated.

  When multiple promoters are present, each promoter may independently be the same or different.

  The 5 ′ untranslated leader sequence can be derived from a promoter selected to express the heterologous gene sequence of the polynucleotide, or can be heterologous to the coding region of the enzyme being generated and is necessary And may be specifically modified to increase the translation of mRNA.

  Termination of transcription is accomplished by a 3 'untranslated DNA sequence that is operably linked to the polynucleotide of interest in an expression vector. The 3 'untranslated region of the recombinant DNA molecule contains a polyadenylation signal that functions to add adenylate nucleotides to the 3' end of RNA in plants. The 3 'untranslated region can be obtained, for example, from various genes that are expressed in plant cells. The nopaline synthase 3 'untranslated region, the 3' untranslated region from the pea small subunit Rubisco gene, and the 3 'untranslated region from the soybean 7S seed storage protein gene are commonly used in this capacity. Also suitable are 3 'transcribed untranslated regions containing the polyadenylation signal of the Agrobacterium tumor-inducible (Ti) plasmid gene.

  Recombinant DNA technology transforms, for example, by manipulating the copy number of polynucleotides in the host cell, the efficiency of transcribing those polynucleotides, the efficiency of translating the resulting transcripts, and the efficiency of post-translational modifications. Can be used to improve the expression of the generated polynucleotide.

  In order to facilitate the identification of transformed cells, the recombinant vector desirably includes a selectable or screenable marker gene as or in addition to the nucleic acid sequence of the polynucleotide as defined herein. A “marker gene” confers a distinctly different phenotype to cells expressing the marker gene, thus allowing differentiation between cells that do not have the marker and such transformed cells It means the gene to make. Selectable marker genes have characteristics that can be “selected” based on resistance to a selection agent (eg, herbicides, antibiotics, radiation, heat, or other treatments that damage non-transformed cells). . Screenable marker genes (or reporter genes) are those features that can be identified through observation or testing, ie by “screening” (eg, β-glucuronidase, luciferase, GFP, or other not present in non-transformed cells). Enzyme activity). For example, as described in US Pat. No. 4,399,216, co-transformation of unlinked genes is also an efficient process in plant transformation, so the marker gene and the nucleotide sequence of interest are There is no need to connect them. The actual selection of the marker is not critical as long as it is functional (ie selective) in combination with a selected cell, such as a plant cell.

  Exemplary selectable markers for selection of plant transformed cells include the hyg gene encoding hygromycin B resistance, the neomycin phosphotransferase (nptII) gene that confers resistance to kanamycin, paramomycin, G418, such as A rat liver-derived glutathione-S-transferase gene that confers resistance to the glutathione-derived herbicides described in European Patent No. EP256223, such as phosphine during overexpression as described in WO 87/05327. Streptomyces viridom chromogenes conferring resistance to glutamine synthetase genes, such as finotricine, glutamine synthetase genes that confer resistance to glutamine synthetase inhibitors, for example, the selective factor phosphinotricin, such as European Patent No. EP275957 Streptomyces viridochromogenes) acetyltransferase gene from, for example, Hinchee et al. (1988), a gene encoding 5-enorshikimate-3-phosphate synthase (EPSPS) that confers resistance to N-phosphonomethylglycine, such as bialaphos described in WO 91/02071 Resistance to the bar gene conferring resistance to bromoxynil (Stalker et al., 1988), nitrilase genes such as bxn derived from Klebsiella ozaenae conferring resistance to bromoxynil (Stalker et al., 1988), methotrexate (Thillet et al., 1988) Confer resistance to dihydrofolate reductase (DHFR) gene, imidazolinone, sulfonylurea, or other ALS-inhibiting chemicals (European Patent No. EP154,204) Examples include, but are not limited to, a mutant acetolactate synthase gene (ALS), a mutant anthranilate synthase gene that confers resistance to 5-methyltryptophan, or a dalapon dehalogenase gene that confers resistance to a herbicide.

  Preferred screenable markers include a uidA gene encoding a β-glucuronidase (GUS) enzyme for which various chromogenic substrates are known, a β-galactosidase gene encoding an enzyme for which the chromogenic substrate is known, calcium sensitivity The aequorin gene (Prasher et al., 1985), the green fluorescent protein gene (Niedz et al., 1995) or derivatives thereof that can be used in bioluminescence detection, or the luciferase (luc) gene that enables bioluminescence detection ( Ow et al., 1986). By “reporter molecule” is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that facilitates determination of promoter activity by reference to a protein product.

  Preferably, the recombinant vector is stably integrated into the genome of a cell such as a plant cell. Thus, a recombinant vector can include appropriate elements that allow the vector to be integrated into the genome or into the chromosome of a cell.

Transfer nucleic acids Transfer nucleic acids can be used to deliver exogenous polynucleotides to cells and can include one, preferably two border sequences, and the polynucleotide of interest. The transfer nucleic acid may or may not encode a selectable marker. Preferably, the transfer nucleic acid forms part of a binary vector in the bacterium, the binary vector further comprising elements that allow replication of the vector in the bacterium, selection or maintenance of bacterial cells containing the binary vector. Upon transfer to a eukaryotic cell, the transferred nucleic acid component of the binary vector can be integrated into the genome of the eukaryotic cell.

  The term “extrachromosomal transfer nucleic acid” as used herein refers to a nucleic acid molecule that can be transferred from a bacterium such as an Agrobacterium species to a eukaryotic cell such as a plant cell. Extrachromosomal transfer nucleic acids are genetic elements well known as elements that can be transferred by subsequent integration of nucleotide sequences contained within their boundaries in the genome of a recipient cell. In this regard, the transfer nucleic acid is typically flanked by two “boundary” sequences, but in some cases a single boundary at one end can be used and the second of the transfer nucleic acid The ends are randomly generated in the transfer process. The polynucleotide of interest is typically located between the left and right border-like sequences of the transfer nucleic acid. The polynucleotide contained within the transfer nucleic acid can be operably linked to a variety of different promoter and terminator regulatory elements that facilitate its expression, ie, transcription and / or translation of the polynucleotide. Transfer DNA (T-DNA) from Agrobacterium species, for example, Agrobacterium tumefaciens or Agrobacterium rhizogenes, and artificial variants / mutants thereof are: Probably the most characterized example of a transfer nucleic acid. Another example is P-DNA containing plant-derived T-DNA border-like sequences (“plant DNA”).

  As used herein, “T-DNA” refers to, for example, an Agrobacterium tumefaciens Ti plasmid or from an Agrobacterium rhizogenes Ri plasmid or an artificial variant thereof that acts as a T-DNA. Refers to the derived T-DNA. T-DNA may contain the entire T-DNA including both right and left border sequences, but only contain the minimal sequence required in cis for transposition, ie, right and T-DNA border sequences is necessary. The T-DNA of the present invention inserted the polynucleotide of interest into them somewhere between the right and left border sequences (if present). Sequences encoding factors required in trans for transfer of T-DNA to plant cells, such as the vir gene, can be inserted into T-DNA or can be present on the same replicon as T-DNA. Or preferably present in trans on a compatible replicon in an Agrobacterium host. Such “binary vector systems” are well known in the art.

  As used herein, “P-DNA” refers to a transfer nucleic acid isolated from a plant genome, or an artificial variant / mutant thereof, at each end or only at one end. Contains DNA border-like sequences. The border-like sequence is preferably at least 50%, at least 60%, at least 70%, and at least 50%, at least 60%, at least 70% 75%, at least 80%, at least 90%, or at least 95% but share less than 100% sequence identity. Thus, P-DNA can be used in place of T-DNA to transfer the nucleotide sequence contained within P-DNA to another cell, eg, derived from Agrobacterium. Prior to insertion of the exogenous polynucleotide to be transferred, the P-DNA can be modified to facilitate cloning and preferably should not encode any protein. The P-DNA is characterized in that it also contains at least a right border sequence and preferably a left border sequence.

  As used herein, a “boundary” sequence of a transfer nucleic acid can be isolated from a selected organism, such as a plant or a bacterium, or can be an artificial variant / mutant thereof. The border sequence can facilitate and facilitate transfer of the polynucleotide and facilitate its integration in the recipient cell genome. In one embodiment, the border sequence is 5-100 base pairs (bp) long, 10-80 bp long, 15-75 bp long, 15-60 bp long, 15-50 bp long, 15 -40 bp length, 15-30 bp length, 16-30 bp length, 20-30 bp length, 21-30 bp length, 22-30 bp length, 23-30 bp length, 24- It is 30 bp long, 25-30 bp long, or 26-30 bp long. Border sequences derived from T-DNA from Agrobacterium species are well known in the art and are described in Lacroix et al. (2008), Tzfila and Citovsky (2006), and Glevin (2003).

  Traditionally, only Agrobacterium species have been used to transfer genes into plant cells, but there are currently a number of identified / developed species that act in a manner similar to Agrobacterium species. A system exists. Some non-Agrobacterium species have been genetically modified in recent years to be competent for gene transfer (Chung et al., 2006, Brootherts et al., 2005). These include Rhizobium sp. NGR234, Sinorhizobium meriroti and Mezorhizobium loti. Bacteria contain a series of virulence genes encoded by T-DNA segments present on bacteria with the mechanisms required for the transformation process, namely the Agrobacterium Ti plasmid, and a separate small binary plasmid. Making it competent for gene transfer. Bacteria manipulated in this way transform different plant tissues (leaf discs, callus and oval tissues), monocotyledonous or dicotyledonous plants, and various different plant species (eg tobacco, rice). Can do.

  As used herein, the terms “transfection”, “transformation” and variations thereof are usually used interchangeably. A “transfected” or “transformed” cell can be a progeny cell that can be engineered or derived from the polynucleotide (s) of interest.

Recombinant cells The present invention also provides recombinant cells, eg, recombinant plant cells that are host cells transformed with one or more polynucleotides or vectors, or combinations thereof, as defined herein. The term “recombinant cell” is used interchangeably herein with the term “transgenic cell”. Suitable cells of the invention include, for example, any cell that can be transformed with a polynucleotide or recombinant vector of the invention that encodes a polypeptide or enzyme described herein. The cell is preferably a cell that can thereby be used to produce lipids. Recombinant cells can be cells in culture, cells in vitro, or cells in organisms such as plants, or in seed or leaf organs, for example. Preferably, the cells are in the plant, more preferably in the seed of the plant, more preferably in the seed of an oilseed plant such as safflower. In one embodiment, the plant cell comprises a lipid or oil having a fatty acid composition as described herein.

  The host cell into which the polynucleotide (s) is introduced can be either an untransformed cell or a cell that has already been transformed with at least one nucleic acid. Such nucleic acids may or may not be associated with lipid synthesis. The host cell of the invention may be capable of endogenously (ie, naturally) producing the polypeptide (s) as defined herein, in which case the recombinant cell derived therefrom is It is possible to produce the polypeptide (s) only after having an enhanced ability to produce the polypeptide (s) or after being transformed with at least one polynucleotide of the invention. possible. In one embodiment, the recombinant cells of the present invention have an enhanced ability to produce nonpolar lipids. The cell can be prokaryotic or eukaryotic. Preferred host cells are yeast, algae, and plant cells. In a preferred embodiment, the plant cell is a seed cell, specifically a cell in a seed cotyledon or endosperm. Examples of algal cells useful as host cells of the present invention include, for example, Chlamydomonas sp. (Eg, Chlamydomonas reinhardtii), Dunaliella sp., Hematocox sp. ), Cholera sp., Thraustochytrium sp., Schizochytrium sp., And Volvox sp.

  The host cell can be an organism suitable for the fermentation process, such as Yarrowia lipolytica or other yeast.

Transgenic plants The present invention also provides a plant comprising an exogenous polynucleotide or polypeptide of the present invention, a cell of the present invention, a vector of the present invention, or a combination thereof. The term “plant” refers to the whole plant, but the term “part thereof” refers to plant organs (eg leaves, stems, roots, flowers, fruits), single cells (eg pollen), seeds, seed parts, eg Refers to embryo, endosperm, scutellum or seed coat, plant tissue, such as vascular tissue, plant cells, and their progeny. Plant parts as used herein include plant cells.

  As used herein, the term “plant” is used in its broadest sense. This includes any herbaceous species, ornamental plants or ornamental plants, crops or cereals (eg oilseed plants, corn, soy), feed or fodder, fruit or vegetable plants, herbaceous plants, woody books, flowers Although a plant or a tree etc. are included, it is not limited to these. It does not mean that the plant is limited to any particular structure. It also refers to unicellular plants (eg, microalgae). The term “parts” with respect to plants refers to plant cells and progeny of the same, multiple plant cells that differentiate mainly into colonies (eg, Volboxes), structures that exist at every stage of plant development, or plant tissue. . Such structures include, but are not limited to, leaves, stems, flowers, fruits, nuts, roots, seeds, seed coats, embryos. The term “plant tissue” refers to differentiated and undifferentiated tissues, including those present in leaves, stems, flowers, fruits, nuts, roots, seeds, eg, embryonic tissues, endosperm, dermal tissues (eg, epidermis, pericytes) ), Vascular tissue (eg, xylem, phloem), or basic tissue (including soft tissue, thick tissue, and / or thick tissue cells), and cells in culture (eg, single cells, protoplasts, callus, Including embryos). The plant tissue may be in planta, organ culture, tissue culture, or cell culture state.

  A “transgenic plant”, “genetically modified plant”, or variant thereof refers to a plant that contains a transgene that is not found in wild-type plants of the same species, variety, or cultivar. A transgenic plant as defined in the context of the present invention has been genetically modified using recombinant technology to result in the production of at least one polypeptide as defined herein in the desired plant or part thereof. Includes plants and their progeny. “Transgenic plant part” has a corresponding meaning.

  The terms “seed” and “grain” are related terms used herein and have overlapping meanings. “Kernel” refers to a mature kernel, eg, a harvested kernel or a kernel that is still in a plant but is ready for harvest, but depending on the context, refers to a kernel after water absorption or germination You can also. Mature kernels generally have a moisture content of less than about 18-20%. “Seeds” includes “growing seeds” as well as “grains” that are mature kernels, but not after absorption or germination. As used herein, a “developing seed” typically refers to a pre-mature seed found in the fertilization structure of a plant after fertilization or flowering, but such pre-mature seed isolated from a plant It can also refer to seeds. Seed development in the in planta method is generally divided into early, intermediate, and late development.

  As used herein, the term “plant storage organ” refers to a part of a plant that specializes in stored energy, for example in the form of proteins, carbohydrates, lipids. Examples of plant storage organs are seeds, fruits, tuberous roots and tubers. The preferred plant storage organ of the present invention is seed.

  As used herein, the term “nutritive tissue” or “nutritive part” or variations thereof are closely related to organs for sexual reproduction of plants, or seeding organs, or flowers, fruits or seeds. Any plant tissue, organ, or part that does not include an associated tissue or organ. The vegetative tissues and parts include at least plant leaves, trunks (including bolts and snails but not heads), tubers and roots, but flowers, pollen, seed coats, embryos and inner Seeds containing endosperm, fruits containing mesocarp, sheaths containing seeds, and heads containing seeds are not included. In one embodiment, the vegetative part of the plant is an aerial plant part. In another or further embodiment, the nutritional part is a green part such as a leaf or stem. Nutritional parts include such parts that primarily provide or support the photosynthetic ability or related function of the plant, or are involved in anchoring the plant.

  As used herein, the term “phenotypically normal” refers to a plant that grows when compared to a genetically modified plant or part thereof, particularly a storage organ, such as an unmodified plant or plant thereof. , Refers to seeds of the present invention whose ability to reproduce has not been significantly reduced. In one embodiment, the phenotypically normal genetically modified plant or part thereof comprises at least one exogenous polynucleotide as defined herein and comprises said polynucleotide (s) Has the ability to grow or reproduce that is essentially identical to the corresponding plant or part not. Preferably, the biomass, growth rate, germination rate, storage organ size, seed size, and / or number of viable seeds produced are such exogenous polynucleotides when grown under the same conditions No less than 90% of plants lacking The term does not encompass plant features that may differ from wild-type plants, but do not give rise to the utility of the plant for commercial purposes.

  Plants provided by the practice of the present invention or contemplated for use in the practice of the present invention include both monocotyledonous and dicotyledonous plants. In preferred embodiments, the plants of the present invention are crop plants (eg, cereals and beans, corn, wheat, potato, tapioca, rice, sorghum, millet, cassava, barley, or peas), or other beans. Plants can be grown for the production of edible roots, tubers, leaves, stems, flowers, or fruits. The plant may be a vegetable or an appreciation plant. Plants of the present invention include safflower (Cartams tinctorius), corn (zeamais), rapeseed (brush kanapus, brush kalapa species), other brassica, for example, rutabaga (brush canapo brassica), mustard (brush kayunsea), ethiopia mustard ( Brush cacarinata (Brassica carinata), hamana (Clam Bear vicinica), camelina (Camelina sativa), sugar beet (Beta ulgaris), clover (Trifolium sp.), Flax (Linham cattle) Tachishimamu), Murasakikimagoya (Medicago sativa), Rice (Oryza sativa), Rye (Secale cerale), Sorghum (Sorghum) Bicolor (Sorghum bicolor), Sorghum vulgare, sunflower (Helianthus anthus), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (solanum) Tuberosum (Solanum tuberosum), Peanuts (Arakis Hippogea), Cottonseed (Gosippium hillstum), Sweet Potato (Lopmoea battus), Cassava (Manihot esculenta, Manihot esculent) (Cofaea genus species), coconut (cocos nufera), pineapple (anana comosus), Tachibana tree (Citrus spp.), Cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), Avocado (Percea America) Persian americana), figs (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olives (Orepapaia) (Carica papaya)), Cashew (Anacardium Occidentale), Macadamia (Macadamia interglifolia), Almond (Prunus amygda) Scan), Ja Toro factory Lucas (Jatropha curcas), lupine, eucalyptus, palm, nuts sage, Pongamia, may be a Oats or barley,.

  Other preferred plants include C4 grasses such as Andropogon gerardi, Bouteloua curtipendula, B. et al. gracilis, Buchloe dactyloides, Panicum virgatum, Schizachyrium scoparium, miscanthus (Miscanthus) species, e.g., Miscanthus x giganteus and Miscanthus sinensis, Sorghastrum nutans, Sporobolus cryptandrus, switch grass (Panicum virgatum), sugar cane (Saccharum officinarum), Brachyaria; C3 grass For example, Elymus canadensis, legumes Lepedeza capitata and Petalostem villosum, broadleaf herb Aster azureus; Rabi and woody plants, such as Quercus ellipsisalis and Q. macrocarpa is included.

  In a preferred embodiment, the plant is an angiosperm.

  In a preferred embodiment, the plant is an oil seed plant, preferably an oil seed crop plant. As used herein, an “oil seed plant” is a plant species used for the commercial production of lipids from plant seeds. “Commercial production” herein means the production of lipids, preferably oils, for sale instead of revenue. The oilseed plant may be rape (such as rapeseed), corn, sunflower, safflower, soybean, sorghum, flax (linseed), or sugar beet. Furthermore, the oilseed plant may be a plant that produces other Brassica, cottonseed, peanuts, poppies, rutabaga, mustard, castor sesame, sesame, safflower, or nuts. Plants can produce high levels of lipids in their fruits, such as olives, oil palm, or coconut. The horticultural plants to which the present invention can be applied are lettuce, endive or vegetable Brassica genus including cabbage, broccoli or cauliflower. The invention can be applied in tobacco, cucurbits, carrots, strawberries, tomatoes or peppers. More preferred plants are oilseed plants in which the developing seeds are non-photosynthetic and are also referred to as “white seed” plants, for example safflower, sunflower, cottonseed, and castor.

In a preferred embodiment, the transgenic plant is homozygous for each and every gene (transgene) introduced so that its progeny do not segregate for the desired phenotype. The transgenic plant may also be heterozygous for the introduced transgene (s), preferably uniformly heterozygous for the transgene, such as for example in the case of F1 progeny grown from hybrid seed. . Such plants can provide advantages such as hybrid strength well known in the art.

Plant Transformation Transgenic plants are generally described in Slater et al. , Plant Biotechnology-The Genetic Manipulation of Plants, Oxford University Press (2003), and Christow and Klee, Handbook of Biotechnology Can be generated using technology.

  As used herein, the terms “stablely transformed”, “stablely transformed” and variations thereof eliminate the need for polynucleotides to actively select for their presence. Refers to the integration of a polynucleotide into the genome of a cell such that it is transferred to progeny cells during cell division. Stable transformed cells or their progeny can be selected and / or identified by any means known in the art, such as Southern blots on chromosomal DNA or in situ hybridization of genomic DNA.

  Since DNA can be introduced into cells in whole plant tissues, plant organs, or explants in tissue culture for transient expression or for stable integration of DNA in the plant cell genome, agro Bacterial mediated transfer is a widely applicable system for introducing genes into plant cells. The use of vectors that incorporate Agrobacterium-mediated plants to introduce DNA into plant cells is well known in the art (eg, US Pat. Nos. 5,177,010, 5,104,310, 5,004863, Or see No. 5159135). The region of DNA to be transferred is limited by the border sequence, and intervening DNA (T-DNA) is usually inserted into the plant genome. Furthermore, T-DNA integration is a relatively rigorous process that results in little rearrangement. In those plant varieties where Agrobacterium-mediated transformation is efficient, it is an optimal method because of the easy and defined nature of gene transfer. Preferred Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing convenient manipulation as described (Klee et al., In: Plant DNA Infectious Agents, Hohn and Schel, eds., Springer-Verlag, New York, pp. 179-203 (1985)).

  Acceleration methods that can be used include, for example, microprojectile bombardment and the like. One example of a method for delivering a transforming nucleic acid molecule to a plant cell is microprojectile bombardment. This method is described in Yang et al. , Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that can be coated with nucleic acids and delivered to cells by propulsion. Such methods are well known in the art. In another embodiment, plastids can be stably transformed. Disclosed methods for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome by homologous recombination (US Pat. No. 5,451,513). No., US Pat. No. 5,545,818, US Pat. No. 5,877,402, US Pat. No. 5,932,479, and International Publication No. WO 99/05265).

  Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. The application of these systems to different plant varieties depends on the ability to regenerate that particular plant line from protoplasts. Exemplary methods for the regeneration of cereals from protoplasts have been described (Fujimura et al., 1985, Toriyama et al., 1986, Abdullah et al., 1986).

  Other methods of cell transformation can also be used, including direct DNA transfer to pollen, direct injection of DNA into plants into plant propagation organs, or cells of immature embryos Incorporation of DNA into plants by, but not limited to, direct injection of DNA into a plant followed by rehydration of dried embryos.

  Plant regeneration, development, and culture from a single plant protoplast or from various transformed explants is well known in the art (Weissbach et al., In: Methods for Plants). Molecular Biology, Academic Press, San Diego, Calif., (1988)). This regeneration and growth process typically includes the steps of selecting transformed cells, culturing those individual cells through the normal embryonic development stage to the rooted seedling stage. Transgenic embryos and seeds are similarly regenerated. Thereafter, the obtained transgenic rooted shoots are planted in an appropriate growth medium such as soil.

  The development or regeneration of plants containing foreign and exogenous genes is well known in the art. Preferably, the regenerated plant is self-pollinated to provide a homozygous transgenic plant. In other cases, pollen obtained from regenerated plants is crossed to plants grown from agronomically important series of seeds. Conversely, pollen from these important families of plants is used to pollinate regenerated plants. The transgenic plants of the present invention containing the desired polynucleotide are cultivated using methods well known to those skilled in the art.

  Methods for transforming dicotyledonous plants, primarily through the use of Agrobacterium tumefaciens, and methods for obtaining transgenic plants are described in cotton (US Pat. No. 5,004,863, US Pat. 159,135, US Pat. No. 5,518,908), soybean (US Pat. No. 5,569,834, US Pat. No. 5,416,011), Brassica (US Pat. No. 5,463,174) No.), peanuts (Cheng et al., 1996), and peas (Grant et al., 1995).

  Methods for transforming cereal plants, such as wheat and barley, and methods for regenerating plants from protoplasts or immature plant embryos to introduce genetic mutations into plants by introduction of exogenous nucleic acids are well known in the art. For example, Canadian Patent No. 2,092,588, Australian Patent No. 61781/94, Australian Patent No. 667939, US Patent No. 6,100,447, International Publication No. PCT / US Patent No. 97/10621 U.S. Pat. No. 5,589,617, U.S. Pat. No. 6,541,257. Renewable wheat cells are preferably derived from immature embryo scutellum, mature embryo, callus derived therefrom, or meristem.

  To confirm the presence of the transgene in transgenic cells and plants, polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Once transgenic plants are obtained, they can be grown to produce plant tissue or parts having the desired phenotype. The plant tissue or plant part may be harvested and / or seeds may be recovered. Seeds can serve as a source for growing additional plants with tissues or parts having the desired characteristics.

  Transgenic plants formed using Agrobacterium or other transformation methods typically contain a single transgenic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the additional gene (s). More preferably, a transgenic plant that is homozygous for the additional gene (s), ie, a transgenic plant that contains two additional genes (one gene at the same locus on each chromosome of a chromosome pair) It is. A homozygous transgenic plant can be obtained by self-fertilizing a hemizygous transgenic plant, germinating a part of the produced seed, and analyzing the obtained plant for the gene of interest.

  Alternatively, two different transgenic plants containing two independently isolated exogenous genes or loci can be crossed (crossed) to produce offspring containing both sets of genes or loci. It should also be understood that it can be done. Proper breeding of the appropriate F1 progeny can generate plants that are homozygous for both the exogenous gene or locus. Backcrossing to the parent plant and outcrossing with non-transgenic plants are also contemplated, such as vegetative propagation. A description of other breeding methods commonly used for different traits and crops is described in Fehr, In: Breeding Methods for Cultivation Development, Wilcox J. et al. ed. , American Society of Agronomy, Madison Wis. (1987).

For safflower transformation, certain useful methods are described by Belide et al. (2011).

TILLING
In one embodiment, TILLING (a target-induced local lesion in the genome) is used, for example, a gene encoding Δ12 desaturase, palmitoyl-ACP thioesterase, ω6 or Δ6 desaturase activity, or two or more thereof Plants can be generated that knock out the combined endogenous genes.

  In the first step, introducing mutations such as new single base pair changes are induced in the plant population by treating the seed (or pollen) with chemical mutagens and then mutating the plants To a generation that is stably inherited. DNA is extracted and seeds from all members of the population are stored, creating a source that can be accessed repeatedly over time.

  For TILLING assays, PCR primers are designed to specifically amplify a single gene target of interest. Specificity is particularly important when the target is a member of a gene family or part of a polyploid genome. The dye product can then be used to amplify the PCR product from pooled DNA from multiple individuals. These PCR products are denatured and re-annealed to allow the formation of mismatched base pairs. Mismatches or heteroduplexes are natural single nucleotide polymorphisms (SNPs) (ie several plants from the population are considered to have the same polymorphism) and induced SNPs (ie very few) Are considered to exhibit mutations). The use of an endonucleotide such as CelI that recognizes and cleaves the mismatched DNA after heteroduplex formation is important for discovering new SNPs within the TILLING population.

Using this approach, thousands of plants can be screened for individuals with a single base pair change in any gene or specific region of the genome, as well as slight insertions or deletions (1-30 bp) It can be specified. The genomic fragment to be assayed can range in size from 0.3 to 1.6 kb. 8x pool, 1.4 kb fragment (not counting the ends of fragments where SNP detection is a problem due to noise) and 96 lanes per assay, this combination allows single up to 1 million base pairs of genomic DNA This makes TILLING a high-throughput approach.
TILLING is further described in Slade and Knauf (2005), and Henikoff et al. (2004).

  In addition to enabling efficient detection of mutations, the high-throughput TILLING approach is also ideal for detecting natural polymorphisms. Thus, probing unidentified homologous DNA by heteroduplexing with a known sequence reveals the number and location of polymorphic sites. Nucleotide changes and minor insertions and deletions are identified, including at least some repeat number polymorphisms. This is called Ecotilling (Comai et al., 2004).

  Each SNP is recorded by its approximate position within a few nucleotides. Therefore, each haplotype can be archived based on its fluidity. Sequence data can be obtained with a relatively slightly increased effort using aliquots of the same amplified DNA used in the mismatch cleavage assay. The left or right sequencing primer for a single reaction is selected by its proximity to the polymorphism. The sequencer software performs multiple alignments and finds base changes identified in the gel bands in each case.

  Ecotilling can be performed cheaper than sequencing the full length, and this method is currently used for most SNP discovery. Rather than mutated plant-derived DNA pools, plates containing aligned ecotype DNA can be screened. Detection is done on a gel with approximately base-pair resolution, and the background pattern is constant across lanes, so bands of the same size can be matched, thus discovering SNPs in a single step, The type can be determined. Thus, due to the fact that an aliquot of the same PCR product used for screening can be subjected to DNA sequencing, the final sequencing of the SNP is simple and efficient.

Mutagenesis Procedures Techniques for generating mutant plant lines are known in the art. Examples of mutagens that can be used to generate mutant plants include irradiation and chemical mutagenesis. Mutants can also be generated by techniques such as T-DNA insertion and transposon-induced mutagenesis. Mutations in any desired gene in plants are site-specific due to artificial zinc finger nuclease (ZFN), TAL effector (TALEN), or CRISPR technology (using Cas9 type nuclease) known in the art. Can be introduced in a conventional manner. The mutagenesis procedure can be performed on any parental cell of the plant, eg, the parental cell in seed or tissue culture.

  Chemical mutagens can be classified by chemical properties, such as alkylating agents, cross-linking agents, and the like. Useful chemical mutagens include N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), procarbazine hydrochloride, chlorambucil, cyclophosphamide, methyl methanesulfonate (MMS). ), Ethyl methanesulfonate (EMS), diethyl sulfate, acrylamide monomer, triethylenemelamine (TEM), melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N′-nitro-nitrosoguanidine (MNNG) ), 7,12 dimethylbenzanthracene (DMBA), ethylene oxide, hexamethylphosphoramide, and bisulphane.

  An example of a suitable irradiation for inducing mutations is that provided by gamma irradiation, for example a cesium 137 source. Gamma irradiation is preferably given to plant cells at a dose of about 60-200 Krad, most preferably at a dose of about 60-90 Krad.

  Plants are typically mutated for a period of time that is sufficient to achieve the desired genetic modification, but insufficient to completely destroy cell viability and the ability of the cells to regenerate into the plant. Exposed to the raw.

  The mutagenesis procedure described above typically results in the generation of a mutant of the gene of interest at a frequency of at least one out of 1000 plants, and screening of a population of mutated plants is subject to any It is meant to be a viable means of identifying gene mutants. Mutant identification can also be achieved by massively parallel nucleotide sequencing techniques.

Marker-based selection Marker-based selection is a widely recognized method of selecting heterozygous plants that are required for backcrossing with recurrent parents in a classical breeding program. The population of plants of each backcross generation is heterozygous for the gene of interest that is usually present in a 1: 1 ratio in the backcross population and uses its molecular markers to distinguish between the two alleles of the gene can do. For example, early selection of plants for further backcrossing while extracting energy from young shoots and testing with specific markers for the introgressed desired trait, concentrating energy and resources on fewer plants Is made. To speed up the backcross program further, instead of fully maturing the seeds, embryos from immature seeds (25 days after flowering) can be cut and grown in nutrient medium under sterile conditions. This process and analysis for the desired genotype, referred to as “embryo rescue”, used in combination with DNA extraction at the trefoil stage, is for further backcrossing to subsequent recurrent parents. Allows rapid selection of plants with the desired traits that can be grown to maturity in the greenhouse or outdoors.

  Any molecular biological technique known in the art that can detect the FAD2, FATB, or FAD6 gene can be used in the methods of the invention. Such methods include the use of nucleic acid amplification, nucleic acid sequencing, nucleic acid hybridization with appropriately labeled probes, single stranded conformational analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), Includes, but is not limited to, heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalytic nucleic acid cleavage, or combinations thereof (see, eg, Lemieux, 2000, Langridge et al., 2001). about). The invention also includes the use of molecular markers to detect polymorphisms linked to alleles of the FAD2, FATB, or FAD6 gene (for example) that give the desired phenotype. Such methods include detection or analysis of restriction fragment length polymorphism (RFLP), RAPD, amplified fragment length polymorphism (AFLP), and microsatellite (simple sequence repeat, SSR) polymorphism. Closely linked markers are described in Langridge et al. (2001) and can be readily obtained by methods well known in the art, such as bulk segregand analysis.

  “Polymerase chain reaction” (“PCR”) consists of a “primer pair” or “primer set” consisting of “upstream” and “downstream” primers, and a catalyst for polymerization, such as a DNA polymerase, typically heat A reaction that makes a replicative copy with a target polynucleotide using a chemically stable polymerase enzyme. Methods for PCR are known in the art and are taught, for example, in “PCR” (Ed. M. J. McPherson and S. G. Moller (2000) BIOS Scientific Publishers Ltd, Oxford). PCR can be performed on cDNA obtained from reverse transcription of mRNA isolated from plant cells. However, if PCR is performed on genomic DNA isolated from plant cells, it will generally be easier.

  A primer is an oligonucleotide sequence that can hybridize to a target sequence in a sequence-specific manner and extend during PCR. An amplicon or PCR product or PCR fragment or amplification product is an extension product that contains primers and a newly synthesized copy of the target sequence. Multiplex PCR systems include multiple sets of primers that result in the simultaneous generation of more than one amplicon. Primers may be perfectly matched to the target sequence or may contain internal mismatch bases that may result in the introduction of restriction enzymes or catalytic nucleic acid recognition / cleavage sites within the specific target sequence. . The primer may also include additional sequences and / or modified or labeled nucleotides to facilitate capture or detection of amplicons. Repeated cycles of heat denaturation of DNA, annealing of primers to complementary sequences, and extension of annealed primers by polymerase result in exponential amplification of the target sequence. The term target or target sequence or template refers to the nucleic acid sequence to be amplified.

  Methods for direct sequencing of nucleotide sequences are well known to those skilled in the art and are described, for example, in Ausubel et al. (See above) and Sambrook et al. (See above). Sequencing can be performed by any suitable method, such as dideoxy sequencing, chemical sequencing, or variations thereof. Direct sequencing has the advantage of determining any base pair variation in a particular sequence.

  Hybridization-based detection systems include, but are not limited to, TaqMan assay and molecular beacon assay (US Pat. No. 5,925,517). The TaqMan assay (US Pat. No. 5,962,233) uses a donor dye at one end and an acceptor dye at the other end so that the dye pair interacts via fluorescence resonance energy transfer (FRET). An allele specific (ASO) probe is used.

In one embodiment, the method described in Example 7 is used in a selection and breeding program to identify and select safflower plants with an ol mutation. For example, the method
i) 5'-ATAAGGCTGTGTCTCAGGGGTT-3 '(SEQ ID NO: 140) and 5'-GCTCAGTTGGGGATACAAGGAT-3' (SEQ ID NO: 141), and ii) 5'-AGTTATGGTTCGATGATCGACG-3 '(SEQ ID NO: 142) and 5'-TTGATGATCG Performing an amplification reaction on genomic DNA obtained from a plant using one or both of the set of primers of (SEQ ID NO: 143).

The term polypeptide "polypeptide" and "protein" are generally used interchangeably.

  A polypeptide or class of polypeptides is defined by a reference amino acid sequence and the degree of identity (% identity) of that amino acid sequence or by one reference amino acid sequence having a greater percent identity than another. obtain. The percent identity of a polypeptide to a reference amino acid sequence is typically determined by GAP analysis (Needleman and Wunsch, 1970, GCG program) with parameters of gap creation penalty = 5, gap extension penalty = 0.3. The The query sequence is at least 100 amino acids long and GAP analysis juxtaposes the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids long and the GAP analysis juxtaposes the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis juxtaposes the two sequences over their entire length. The polypeptide or polypeptides may have the same enzymatic activity or different activity as the reference polypeptide, or may lack that activity. Preferably, the polypeptide has an enzyme activity that is at least 10% of the activity of the reference polypeptide.

  As used herein, a “biologically active fragment” is a composition of the invention that maintains a defined activity of a full-length reference polypeptide, eg, a Δ12 desaturase, palmitoyl-ACP thioesterase, or Δ6 desaturase activity. Part of the polypeptide. Biologically active fragments as used herein do not include full-length polypeptides. Biologically active fragments can be portions of any size as long as they maintain the defined activity. Preferably, the biologically active fragment retains at least 10% of the activity of the full length polypeptide.

  It will be appreciated that a percent identity value higher than that provided herein for a defined polypeptide or enzyme encompasses preferred embodiments. Thus, where applicable, the polynucleotide / enzyme is at least 50%, more preferably at least 60%, more preferably at least 65%, more than the associated designated SEQ ID NO, considering the minimum% identity number Preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably Is at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably Less 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%. More preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9%.

  Mutants of the amino acid sequence of a polypeptide as defined herein can be prepared by introducing an appropriate nucleic acid into a nucleic acid as defined herein or by in vitro synthesis of the desired polypeptide. Such mutations include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. Combinations of deletions, insertions, or substitutions can be made to arrive at the final construct, provided that the final polypeptide product has the desired characteristics.

  Mutant (altered) polypeptides can be prepared using any technique known in the art, for example, using directed evolution or rationale design strategies (see below). ). To determine whether a product derived from mutated / modified DNA has or lacks Δ12 desaturase, palmitoyl-ACP thioesterase, or ω6Δ6 desaturase activity, the techniques described herein are used. And can be easily screened.

  In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on the feature (s) to be modified. The site of the mutation can be identified, for example, by (1) first substituting with a conserved amino acid selection and then substituting with a significantly different selection depending on the result achieved (2) It can be modified individually or sequentially by deleting, or (3) by inserting other residues adjacent to the located site. One skilled in the art will appreciate that the use of non-conservative substitutions can be used to generate mutations with reduced enzyme activity.

  Amino acid sequence deletions are generally about 1 to 15 residues, more preferably about 1 to 10 residues, typically about 1 to 5 contiguous residues, although mutations are particularly active Can be longer if designed to mitigate.

  Substitution mutants have at least one amino acid residue in the removed polypeptide and a different residue inserted in its place. Sites of greatest interest for substitutional mutagenesis to inactivate enzymes include those identified as active site (s). Other sites of interest are sites where certain residues from different strains or species are identical. These positions can be important for biological activity. Conservative substitutions are shown in Table 1, but non-conservative substitutions are substitutions that are not conservative substitutions.

  In one embodiment, the polypeptide of the invention is a Δ12 desaturase, a sequence provided in any one of SEQ ID NOs: 27-34, 36, or 37, a biologically active fragment thereof, or An amino acid having an amino acid sequence that is at least 40% identical to any one of SEQ ID NOs: 27-34, 36, or 37.

  In another embodiment, the polypeptide of the present invention is an oleate Δ12 desaturase present in the seed of an oil seed plant (oil seed) and is any one of SEQ ID NOs: 27, 28, or 36. Or a biologically active fragment thereof, or an amino acid having an amino acid sequence that is at least 40% identical to any one of SEQ ID NOs: 27, 28, or 36.

  In another embodiment, the polypeptide of the invention is a Δ12-acetylenase and the sequence provided in SEQ ID NO: 37, a biologically active fragment thereof, or an amino acid sequence that is at least 40% identical to SEQ ID NO: 37 An amino acid having

  In another embodiment, a polypeptide of the invention is a palmitoleate Δ12 desaturase and is at least 40% identical to the sequence provided in SEQ ID NO: 35, a biologically active fragment thereof, or SEQ ID NO: 35. Includes amino acids having an amino acid sequence.

In another embodiment, the polypeptide of the invention is palmitoyl-ACP thioesterase (FATB), the sequence provided in any one of SEQ ID NOs: 44 or 45, a biologically active fragment thereof, or It comprises an amino acid having an amino acid sequence that is at least 40% identical to any one of SEQ ID NOs: 44 or 45.

  In another embodiment, the polypeptide of the present invention is palmitoyl-ACP thioesterase (FATB) present in the seeds of oilseed plants, the sequence provided in SEQ ID NO: 45, its biologically active A fragment, or an amino acid having an amino acid sequence that is at least 40% identical to SEQ ID NO: 45.

  In another embodiment, a polypeptide of the invention is a Δ6 desaturase and has a sequence provided in SEQ ID NO: 48, a biologically active fragment thereof, or an amino acid sequence that is at least 40% identical to SEQ ID NO: 48. Including amino acids.

  Preferred features of the enzymes of the present invention are provided in the Example section, specifically Example 2, for safflower FAD2.

  The polypeptides described herein can be expressed as a fusion to at least one other polypeptide. In a preferred embodiment, the at least one other polypeptide is selected from the group consisting of a polypeptide that improves the stability of the fusion protein and a polypeptide that aids in purification of the fusion protein.

Production of lipids and / or oils high in oleic acid Techniques commonly practiced in the art can be used to extract, process, purify, and analyze the lipids produced by the plants of the present invention, particularly seeds. . Such techniques are described in Feliidoon Shahidi, Current Protocols in Food Analytical Chemistry, John Wiley & Sons, Inc. (2001) D1.1.1-D1.1.1.11 and Perez-Vich et al. (1998) etc. are described and explained over the source literature.

Seed Oil Production Typically, plant seeds are cooked, pressed and / or extracted to produce crude seed oil, which is then degummed, refined, bleached and deodorized. In general, techniques for grinding seeds are known in the art. For example, oil seeds can be sliced using a smooth roller with a moisture content of, for example, 8.5% and a gap setting of 0.23 to 0.27 mm. Depending on the type of seed, water may not be added before grinding. Heating inactivates the enzyme, facilitates further cell disruption, coalesces lipid droplets and aggregates protein particles, all of which facilitate the extraction process.

  In one embodiment, most of the seed oil is released by passage through a screw press. The cake spouted from the screw press is then solvent extracted, for example with hexane, using a heat tracking column. Alternatively, the crude seed oil produced by the pressing operation can be passed through a sedimentation tank having a grooved wire drainage top to remove solids that are expressed with the seed oil during the pressing operation. After removal of hexane, the solid residue from pressing and extraction is seed powder, which is usually used as animal food. The purified seed oil can be passed through a plate and frame filter to remove any remaining solid particulates. If desired, the seed oil recovered from the extraction process can be combined with purified seed oil to produce a mixed crude seed oil.

  Once the solvent is removed from the crude seed oil, the pressed and extracted portions are combined and subjected to conventional lipid treatment techniques such as degumming, caustic purification, bleaching, and deodorization. Some or all of the steps may be omitted, for example depending on the characteristics of the production pathway for feed oil, but limited application may be required for oleochemical applications Further purification steps are required.

Degumming Degumming is an early step in oil refining and its main purpose is the removal of most of the phospholipids from the oil, which is expressed as about 1-2% of the total extracted lipids. obtain. At 70-80 ° C., the addition of about 2% water, typically containing phosphoric acid, to the crude oil results in the separation of most of the phospholipids, as well as trace metals and pigments. The insoluble material that is removed is primarily a mixture of phospholipids and triacylglycerols, also known as lecithin. By adding concentrated phosphoric acid to the crude seed oil, degumming can be performed to convert non-hydratable phospholipids to a hydrated form and to chelate the trace metals present. The gum is separated from the seed oil by centrifugation.

Alkaline refining Alkaline refining is one of the refining processes for treating crude oil and is sometimes referred to as neutralization. It usually follows degumming and before decolorization. After degumming, the seed oil can be treated by adding a sufficient amount of alkaline solution to titrate all of the fatty acids and phosphoric acid, thus removing the soap that is formed. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate, and ammonium hydroxide. This process is typically performed at room temperature to remove the free fatty acid fraction. The soap is removed by centrifugation or by extraction into a solvent for the soap, and the neutralized oil is washed with water. If necessary, any excess alkali in the oil can be neutralized with a suitable acid such as hydrochloric acid or sulfuric acid.

Bleaching is carried out at 90-120 ° C. in the presence of bleaching earth (0.2-2.0%) and in the absence of oxygen by the oil being operated with nitrogen or steam or under vacuum. Purification process heated for ~ 30 minutes. This step in oil processing is designed to remove unwanted pigments (carotenod, chlorophyll, gossypol, etc.), and this process also removes traces of oxidation products, trace metals, sulfur compounds, and soaps.

Deodorization Deodorization is the treatment of oils and fats at high temperatures (200-260 ° C.) and low pressures (0.1-1 mm Hg). This is typically achieved by introducing steam into the seed oil at a rate of about 0.1 ml / min / 100 ml (seed oil). About 30 minutes after application, the seed oil is allowed to cool under vacuum. Seed oil was typically transferred to a glass container and aerated with argon before being stored under cooling. This treatment improves the color of the seed oil and removes most of the volatile or odorous compounds, including any remaining free fatty acids, monoacylglycerols, and oxidation products.

Dewaxing Dewaxing is the commercialization of oils for the separation of oils and fats into solid (stearin) and liquid (olein) fractions, sometimes by crystallization at sub-ambient temperature. It is a process used in industrial production. It was originally applied to cottonseed oil to produce a product with no solids. It is typically used to reduce the saturated fatty acid content of the oil.

Transesterification Reaction The transesterification reaction is the process of exchanging fatty acids in and between TAGs by first releasing the fatty acids from the TAG as free fatty acids or as fatty acid esters, usually fatty acid ethyl esters. When combined with the fractionation process, transesterification can be used to modify the fatty acid composition of lipids (Marangoni et al., 1995). The transesterification reaction can use either chemical or enzymatic methods, the latter being position specific (sn-1 / 3 or sn-2 specific) for fatty acids on the TAG. Use the resulting lipase or have selectivity for some fatty acids over others (Speranza et al, 2012). Fatty acid fractions that increase the concentration of LC-PUFA in the oil can be obtained by, for example, freezing crystallization, complex formation with urea, molecular distillation, supercritical fluid extraction, and silver ion complexation. This can be accomplished by any of the methods known in the art. Complex formation with urea is a preferred method for its simplicity and efficiency in reducing the levels of saturated and monounsaturated fatty acids in oil (Gamez et al., 2003). First, the oil TAG splits the fatty acids into their components, often in the form of fatty acid esters, by hydrolysis or by lipase, and these free fatty acids or fatty acid esters are used for complex formation. And mixing with ethanol solution of urea. Saturated and monounsaturated fatty acids can be easily synthesized with urea, cooled, crystallized and then removed by filtration. The non-urea complexed fraction is thereby enriched with LC-PUFA.

Hydrogenation Fatty acid hydrogenation typically involves treatment with hydrogen in the presence of a catalyst. Non-catalytic hydrogenation takes place only at very high temperatures.

  Hydrogenation is commonly used in the processing of vegetable oils. Hydrogenation converts unsaturated fatty acids to saturated fatty acids and, in some cases, trans fats. Hydrogenation results in the conversion of liquid vegetable oils to solid or semi-solid fats such as those present in margarine. Changing fat saturation changes some important physical properties, such as melting range, so that liquid oils become semi-solid. Solid or semi-solid fats are preferred for baking because the fat is mixed with flour to provide a more desirable feel in the baked product. Partially hydrogenated vegetable oils are cheaper than animal source fats, are available in a wide range of consistency, and have other desirable characteristics (eg, increased oxidative stability / longer shelf life). And they are the main fats used as shortening in most commercial baked goods.

  In one embodiment, the lipid / oil of the present invention is not hydrogenated. What indicates that the lipid or oil is not hydrogenated is the absence of any trans fatty acids in the TAG.

Lipid Use Lipids produced by the methods described herein, such as seed oil, preferably safflower seed oil, have a variety of uses. In some embodiments, the lipid is used as an edible oil. In other embodiments, the lipids are purified and used as a lubricating oil or for other industrial applications such as the synthesis of plastic products. It can be used in the manufacture of cosmetics, soaps, softeners, electrical insulation or synthetic detergents. It can be used to produce agricultural chemicals such as surfactants or emulsifiers. In some embodiments, the lipid is purified to produce biodiesel. The oils of the present invention can be advantageously used in paints or varnishes, since the absence of linolenic acid means that they do not readily discolor.

  Industrial products produced using the process of the present invention are hydrocarbon products such as fatty acid esters, preferably fatty acid methyl esters, and / or fatty acid ethyl esters, alkanes such as methane, ethane, or long chains. Alkanes, mixtures of long chain alkanes, alkenes, biofuels, carbon monoxide and / or hydrogen gas, bioalcohols such as ethanol, propanol or butanol, biochar, or a combination of carbon monoxide, hydrogen and biochar possible. The industrial product is a mixture of any of these components, for example alkanes, or mixtures of alkanes and alkenes, preferably (> 50%) C4-C8 alkanes, or mainly C6-C10. It can be an alkane, or a mixture that is primarily a C6-C8 alkane. Industrial products are neither carbon dioxide nor water, but these molecules can be produced in combination with industrial products. The industrial product can be a gas at atmospheric pressure / room temperature, or preferably a liquid, or a solid such as biochar, or the process can be a gas component, a liquid component, and a solid component such as carbon monoxide, It can be produced in combinations such as hydrogen gas, alkanes and biochar and then separated. In one embodiment, the hydrocarbon product is primarily fatty acid methyl esters. In an alternative embodiment, the hydrocarbon product is a product other than a fatty acid methyl ester.

  Heating can be applied to the process, for example, by pyrolysis, combustion, gasification, or with enzymatic digestion (including anaerobic digestion, composting, fermentation). Low temperature gasification is performed at about 700 degreeC-about 1000 degreeC, for example. High temperature gasification is performed at about 1200 to about 1600 degreeC, for example. Low temperature pyrolysis (slow pyrolysis) is performed at about 400 ° C, while high temperature pyrolysis is performed at about 500 ° C. Mesophilic digestion is performed at about 20 ° C to about 40 ° C. Hot digestion is performed at about 50 ° C to about 65 ° C.

  Chemical means include, but are not limited to, catalytic cracking, anaerobic digestion, digestion, fermentation, composting, and transesterification. In one embodiment, the chemical means uses a catalyst or mixture of catalysts that can be applied with heating. This process may use homogeneous catalysts, heterogeneous catalysts, and / or enzyme catalysts. In one embodiment, the catalyst is a transition metal catalyst, molecular sieve type catalyst, activated alumina catalyst, or sodium carbonate as the catalyst. The catalyst includes an acid catalyst, such as sulfuric acid, or an alkali catalyst, such as potassium hydroxide or sodium hydroxide, or other hydroxide. Chemical means can include transesterification of fatty acids in lipids, and the process can use homogeneous, heterogeneous, and / or enzymatic catalysis. The transformation can include pyrolysis where heat is applied and chemical means can be applied, and a transition metal catalyst, molecular sieve type catalyst, activated alumina catalyst, or sodium carbonate as the catalyst can be used.

  Enzymatic means include, but are not limited to, anaerobic digestion, digestion, fermentation, digestion with microorganisms in composting, or digestion with recombinant enzyme proteins.

Biofuel The term “biofuel” as used herein includes biodiesel and bioalcohol. Biodiesel can be made from oils derived from plants, algae, and fungi. Bioalcohol is produced from sugar fermentation. The sugar can be extracted directly from plants (eg, sugarcane), derived from plant starch (eg, corn or wheat), or made from cellulose (eg, trees, leaves, or stems).

  Biofuels are currently more expensive to produce than petroleum-based fuels. In addition to processing costs, biofuel grains require planting, fertilization, pesticide and herbicide use, harvesting, and transportation. The plants, algae, and fungi of the present invention can reduce biofuel costs.

  General methods for the production of biofuels are described, for example, by Maher and Bressler (2006), Maher and Bressler (2007), Greenwell et al. (2011), Karmakar et al. (2010), Alonso et al. (2010), Lee and Mohamed (2010), Liu et al. (2010), Gong and Jiang (2011), Endalew et al. (2011), and Semwal et al. (2011).

Biodiesel The production of biodiesel or alkyl esters is well known. There are three basic pathways for ester formation from lipids: 1) base-catalyzed transesterification of lipids with alcohols, 2) direct oxidation-catalyzed transesterification of lipids with methanol, and 3) lipids to fatty acids. Conversion followed by acid catalysis to alkyl ester.

  Any method for preparing fatty acid alkyl esters and glyceryl ethers (one, two, or three of the hydroxy groups in glycerol are etherified) can be used. For example, fatty acids can be prepared, for example, by hydrolyzing or saponifying triglycerides, respectively, using acid or base catalysts, or by using enzymes such as lipases or esterases. Fatty acid alkyl esters can be prepared by reacting a fatty acid with an alcohol in the presence of an acid catalyst. Fatty acid alkyl esters can also be prepared by reacting triglycerides with alcohols in the presence of acid or base catalysts. Glycerol ethers can be prepared, for example, by reacting glycerol with an alkyl halide in the presence of a base or with an olefin or alcohol in the presence of an acid catalyst.

  In some preferred embodiments, the lipids are transesterified to produce methyl esters and glycerol. In some preferred embodiments, the lipid is reacted with an alcohol (eg, methanol or ethanol) in the presence of a catalyst (eg, potassium hydroxide or sodium hydroxide) to produce an alkyl ester. Alkyl esters can be used for biodiesel or can be mixed with petroleum-based fuels.

  The alkyl ester can be mixed directly with the diesel fuel or it can be washed with water or other nest solution prior to mixing to remove various impurities including the catalyst. It is possible to neutralize the acid catalyst with a base. However, this process produces a salt. To avoid engine corrosion, it is preferred to minimize salt concentration in the fuel additive composition. The salt can then be removed from the composition, for example, by washing the composition with water.

  In another embodiment, the composition is dried after it has been washed, for example by passing the composition through a desiccant such as calcium sulfate.

  In yet another embodiment, neutral fuel addition is achieved by using a polymeric acid, such as Dowex 50 ™, which is a resin containing sulfonic acid groups, without generating a salt or using a wash step. Get things. The catalyst is easily removed by filtration after the esterification and etherification reactions are complete.

Plant Triacylglycerol as a Biofuel Source The use of plant triacylglycerol for the production of biofuel is described in Durrett et al. (2008). Briefly, vegetable oils consist primarily of various triacylglycerols (TAGs) and the molecule consists of three fatty acid chains (usually 18 or 16 carbons long) esterified to glycerol. . Fatty acyl chains are chemically similar to the aliphatic hydrocarbons that make up most of the molecules found in gasoline and diesel. The hydrocarbons in gasoline contain 5-12 carbon atoms per molecule, and this volatile fuel is mixed with air and ignited with sparks in a conventional engine. In contrast, diesel fuel components typically have 10-15 carbon atoms per molecule and are ignited by the very high compression obtained in diesel engines. However, most plant TAGs are much higher than the viscosity range of conventional diesel, which is 17.3-32.9 mm ² s ^-1 , respectively, compared to 1.9-4.1 mm ² s ^-1. Has a viscosity range (ASTM D975, Knothe and Steidley, 2005). This high viscosity results in poor fuel atomization in modern diesel engines and causes problems resulting from incomplete combustion such as carbon deposition and coking (Ryan et al., 1984). To overcome this problem, TAG is converted to a lower viscosity fatty acid ester by esterification with a primary alcohol, usually methanol. The resulting fuel is usually referred to as biodiesel and has a dynamic viscosity of 1.9 to 6.0 mm ² s ⁻¹ (ASTM D6751). Fatty acid methyl esters (FAME) found in biodiesel have a high energy density, which is reflected by the high heat of combustion that is similar if not as high as that of conventional diesel (Knothe, 2005). Similarly, the cetane number of FAME found in biodiesel (a measure of diesel ignition) exceeds that of conventional diesel (Knothe, 2005).

  Vegetable oil is mainly composed of five common fatty acids: palmitate (16: 0), stearate (18: 0), oleate (18: 1), linoleate (18: 2). , And linolenate (18: 3), but longer or shorter fatty acids may also be the major component, depending on the particular species. These fatty acids differ from each other in terms of acyl chain length and double bond, resulting in different physical properties. Therefore, the fuel properties of biodiesel derived from a mixture of fatty acids depends on its composition. Therefore, modification of the fatty acid profile can improve biodiesel fuel properties such as low temperature fluidity, oxidation stability, and NOx release. Altering the fatty acid composition of the TAG can reduce the viscosity of the vegetable oil and eliminate the need for chemical modification, thus improving the cost effectiveness of the biofuel.

Feed The lipid / oil of the present invention has its very high oleic acid content and low levels of linoleic acid (less than 3.2%), saturated fatty acids such as palmitic acid, and essentially zero levels of linolenic acid. Therefore, it has an advantage for food use. This provides an oil with high oxidative stability that makes food applications that require heating ideal, such as fried applications such as fried potatoes, with low acid odor. The oil has a high OSI (Oxidation Stability Index) measured as the length of time that the oil can be held at 110 ° C., for example longer than 20 or 25 hours, preferably longer than 30 hours or longer than 50 hours. Have. Low levels of saturated fatty acids provide health benefits compared to other vegetable oils, as saturated fatty acids are associated with adverse health effects. These oils are also desirable in some markets because they inherently have a zero trans fatty acid content and the trans fatty acids are associated with adverse heart health effects or raise LDL cholesterol. In addition, due to its very low level of polyunsaturated fatty acids, these oils reduce the level of PUFA and do not require hydrogenation to produce trans fatty acids. These oils are also advantageous in reducing the onset or severity of obesity and diabetes. They are also desirable for food applications in that they contain only natural fatty acids (Scarth and Tang, 2006).

  For the purposes of the present invention, “feed”, when taken into the body, (1) nourishes or increases tissue or shares energy and / or (2) suitable Includes any food or preparation for human or animal consumption (including enteral and / or parenteral intake) that maintains, restores, or supports nutritional status or metabolic function. The feed of the present invention comprises a nutritional composition for infants and / or infants.

  The feed of the present invention may be, for example, a cell of the present invention, a plant of the present invention, a plant part of the present invention, a seed of the present invention, an extract of the present invention, a product of the method of the present invention, or a suitable carrier (s). Composition). The term “carrier” is used in its broadest sense to encompass any ingredient that may or may not have nutritional value. As will be appreciated by those skilled in the art, the carrier must be suitable (or used at a sufficiently low concentration) for use in the feed so that it does not adversely affect the organisms that feed the feed.

  The feed of the present invention comprises lipids produced directly or indirectly by use of the methods, cells, or organisms disclosed herein. The composition may be in solid or liquid form. In addition, the composition may include edible macronutrients, vitamins, and / or minerals in amounts desired for a particular use. The amount of these or other ingredients depends on whether the composition is intended for use by normal individuals or for individuals with special needs, such as those suffering from metabolic disorders, etc. Change.

  A food product can be produced by mixing oil with one or more other ingredients such that the food product contains oil, or mixed with one or more other ingredients such as salad dressing or mayonnaise Food additives can be made. The food or food additive may comprise 1 wt% to 10 wt% or more oil. The oil can be mixed with other vegetable oils to provide an optimal composition, or solid oil, or coconut oil to provide a semi-solid shortening. Foods or food additives generated from oil include salad dressings, mayonnaise, margarines, breads, cakes, biscuits (cookies), croissants, baked goods, pancakes or pancake mixes, custards, frozen desserts, non-dairy foods It is.

  Examples of suitable nutritive carriers include, but are not limited to, macronutrients such as edible fats, carbohydrates, and proteins. Examples of such edible fats include, but are not limited to, coconut oil, borage oil, fungal oil, black current oil, soybean oil, and mono- and di-glycerides. Examples of such carbohydrates include, but are not limited to, glucose, edible lactose, and hydrolyzed starch. In addition, examples of proteins that can be utilized in the nutritional compositions of the present invention include soy protein, electrodialyzed whey, electrodialyzed skim milk, or hydrolysates of these proteins. It is not limited.

  With respect to vitamins and minerals, calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C, and B complexes are fed into the feed of the present invention. It may be added to the composition. Other such vitamins and minerals may also be added.

  The components utilized in the feed composition of the present invention can be derived from semi-purified or purified. Semi-purified or purified means material prepared by purification of natural materials.

  The feed composition of the present invention may be added to foods even when dietary supplementation is not required. For example, the composition may include any type of margarine, modified butter, cheese, milk, yogurt, chocolate, candy, snack, salad oil, cooking oil, cooking fat, meat, fish, and beverages. It may be added to food.

  In addition, lipids produced by the present invention, or host cells transformed to contain and express a gene of interest can also be used to ingest animal tissue, or milk fatty acid composition or egg fatty acid composition for human or animal consumption. Alternatively, it may be used as an animal food supplement to modify it to be more desirable for animal health and well-being. Examples of such animals include pets such as sheep, cows, horses, dogs and cats.

  Furthermore, the feed of the present invention can be used in aquaculture to increase the level of fatty acids in fish for human or animal consumption.

  Preferred feeds of the present invention are plants, seeds and other plant parts, such as leaves, fruits and stems, which can be used directly as food or diet for humans or other animals. For example, the animals can directly eat such plants grown in the field or can be fed a higher measured amount in a controlled feeding.

Compositions The present invention also encompasses compositions, particularly pharmaceutical compositions, comprising one or more lipids or oils produced using the methods of the present invention.

  The pharmaceutical composition comprises standard well known non-toxic pharmaceutically acceptable carriers, adjuvants, or vehicles such as phosphate buffered saline, water, ethanol, polyols, vegetable oils, wetting agents, Alternatively, one or more of the lipids may be included in combination with an emulsifier such as a water / oil emulsifier. The composition may be in liquid or solid form. For example, the composition may be in the form of a tablet, capsule, ingestible liquid, powder, topical ointment, or cream. The proper fluidity can be maintained, for example, by maintaining the required particle size in the case of dispersion and by the use of surfactants. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. In addition to such inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

  Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, or these A mixture of these substances may also be included.

  Solid dosage forms such as tablets and capsules can be prepared using techniques well known in the art. For example, lipids produced according to the present invention may be combined with binders such as acacia, corn starch, or gelatin, disintegrants such as potato starch or starch using conventional tablet bases such as lactose, sucrose, and corn starch. Tablets can be combined with alginic acid and lubricants such as stearic acid or magnesium stearate. Capsules can be prepared by incorporating these excipients into gelatin capsules together with antioxidants and related lipid (s).

  For intravenous administration, lipids produced by the present invention or their derivatives may be incorporated into commercial formulations.

  Typical doses of certain fatty acids are 0.1 mg to 20 g taken 1 to 5 times per day (up to 100 g per day), preferably about 10 mg to about 1, 2, 5, Or within the range of 10 g (taken in single or multiple doses). As is known in the art, a minimum of about 300 mg / day of fatty acid is desirable. However, it will be appreciated that any amount of fatty acid is beneficial to the subject.

  Possible routes of administration of the pharmaceutical composition of the present invention include, for example, enteral and parenteral. For example, liquid preparations may be administered orally. In addition, a homogeneous mixture can be completely dispersed in water and mixed with physiologically acceptable diluents, preservatives, buffers, or propellants under sterile conditions to form sprays or inhalants. it can.

  The dose of the composition to be administered to the subject can be determined by one skilled in the art and depends on various factors such as the subject's weight, age, overall health, medical history, immune status, and the like.

  Furthermore, the compositions of the present invention can be utilized for cosmetic purposes. The composition can be added to an existing cosmetic composition so that a mixture is formed or the fatty acids produced according to the invention can be used as the only “active” ingredient in the cosmetic composition. .

Example 1. General materials and methods
Plant material and growth conditions Safflower plants (Kartams tinctorius) genotype SU, S-317, S-517, LeSaf496, CW99-OL, and Ciano-OL were treated for 16 hours (25 ° C.) / 8 hours (22 ° C. ) From seeds in a greenhouse in a potting mixture of pearlite and sand loam under a day / night cycle. Wild-type variant SU, a high linoleic acid variant, was obtained from NSW's Heffernan Seeds. PI603208 (LeSaf496, ATC120562) and CW99-OL (ATC120561) seeds were obtained from the Australian Temperate Field Crops Collection.

  Plant tissues for DNA and RNA extraction including leaves, roots, cotyledons and hypocotyls were harvested from safflower seedlings 10 days after germination. The flowering head is obtained on the first day when the flower opens, and the developing embryo is divided into three parts after flowering (DPA) 7 days (early), 15 days (middle), and 20 days (late). Harvested at the developmental stage. Samples were immediately cooled in liquid nitrogen and stored at −80 ° C. until DNA and RNA extraction was performed.

  The safflower florets are tubular and are usually self-pollinated, usually in less than 10% outcross (Knowles 1969). Insects, not wind, can increase the level of self-pollination in the field. Non-pollinated stigmas can be acceptable for several days. Each flower head (safflower head) contains about 15-30 fruit. The seed mass of developing seeds in plants increases rapidly during the first 15 days after flowering. The oil content increases 5 to 10 times during the period of 10-15 DAP, reaching the highest level at about 28 DPA (Hill and Knowles 1968). When safflower seeds and plants mature physiologically about 5 weeks after flowering, most of the leaves turn brown and a slightly greenish color remains on the leaves of the slowest flowering head The seeds are in a state of harvesting. Seeds were easily harvested by rubbing the heads by hand.

Lipid Analysis Isolation of Lipid Samples from Single Seeds for Rapid Fatty Acid Composition Analysis After harvesting at plant maturity, safflower seeds are allowed to reach room temperature for 3 days at 37 ° C. and then at room temperature if not immediately analyzed. The seeds were dried by storing. Single seeds or pooled seeds were ground between small filter papers and the exuded seed oil soaked into the paper was analyzed for fatty acid composition by the GC method described below.

Isolation of total lipids from post-emergent hemicotyledons For example, for screening against progeny seeds from transgenic plants, safflower seeds were germinated on moist filter paper in Petri dishes for 1 day. Cotyledons were carefully removed from each germinated seed for lipid analysis. The rest of each seedling was transferred to soil and the resulting plants were grown to maturity and harvested seeds that maintained the transgenic lines.

Extraction of oil from seeds using a Soxhlet apparatus For quantitative extraction of seed oil, harvested safflower seeds were dried in an oven at 105 ° C. overnight and ground in Puck Mill for 1 minute. Ground seed material (about 250 grams) was collected in a pre-weighed thimble and weighed before oil extraction. After the addition of one layer of cotton wool on the metal, the oil was first extracted with solvent (Petoleum Spirit 40-60C) in a Sockley apparatus at 70-80 ° C. The mixture was then refluxed overnight with solvent siphoned into the extraction flask every 15-20 minutes. The dissolved and extracted oil was recovered by evaporation from the solvent using a rotary evaporator under vacuum. The extracted oil was weighed to determine the oil content. A small aliquot was diluted in chloroform and analyzed by gas chromatography to determine the fatty acid composition of the extracted oil.

Isolation of total lipids from leaf material Leaf tissue samples were lyophilized and weighed, and total lipids were extracted from approximately 10 mg dry weight samples as described by Bligh and Dyer (1959).

Separation of lipids If necessary, the TAG fraction is separated from other lipid components using a two-phase thin film chromatography (TLC) system on pre-coated silica gel plates (Silica gel 60, Merck). did. Extracted lipid samples corresponding to 10 mg dry weight of leaf tissue were used to remove non-polar wax using hexane / diethyl ether (98/2 v / v) in the first phase and then the second phase. And chromatographed using hexane / diethyl ether / acetic acid (70/30/1 v / v / v). Where necessary, chloroform / methanol / water (65/25/4 v / v / v) in the first direction and chloroform / methanol / NH ₄ OH / ethylpropyl in the second direction. Extracted from an equivalent of 5 mg dry weight leaf equivalent using 2D TLC (Silica gel 60, Merck) using amine (130/70/10/1 v / v / v / v) In the prepared lipid sample, polar lipids were separated from nonpolar lipids. Lipid spots and appropriate standards run on the same TLC plate were visualized by short-term exposure to iodine vapor, collected in vials, and transmethylated for GC analysis to produce FAME as follows. .

Preparation of Fatty Acid Methyl Ester (FAME) and Gas Chromatography (GC) Analysis For fatty acid composition analysis by GC, the extracted lipid sample prepared as described above was transferred to a glass tube and placed in 2 mL of methanol (Supelco). Transmethylated in 1M HCl at 80 ° C. for 3 hours. After cooling to room temperature, 1.3 mL of 0.9% NaCl and 800 μL of hexane were added to each tube and FAME was extracted into the hexane phase. To determine the fatty acid composition, the temperature increase program was changed to an initial temperature of 150 ° C., maintained for 1 minute, increased to 210 ° C. at 3 ° C./minute, then increased to 240 ° C. at 50 ° C./minute, and finally In general, Zhou et al. The FAMEs were separated by gas chromatography (GC) using an Agilent Technologies 7890A gas chromatograph (Palo Alto, California, USA) equipped with a 30m BPX70 column, essentially as described by (2011). Peaks were quantified using Agilent Technologies ChemStation software (Rev B.03.01 (317), Palo Alto, California, USA). The peak response is the authentic Nu-Check GLC standard- containing the same ratio of 31 different fatty acid methyl esters including 18: 1, 18: 0, 20: 0, and 22: 0 used for calibration. 411 (Nu-Check Prep Inc, MN, USA). The ratio of each fatty acid in the sample was calculated based on the individual and total peak areas in the fatty acid.

Analysis of FAME by gas chromatography (mass spectrometry) Modification of 2,4-dimethyloxazoline (DMOX) and confirmation of the position of the double bond in FAME by GC-MS analysis were performed using Shimadzu GC-MS equipped with a 30m BPX70 column. The procedure was as described above (Zhou et al., 2011) except that QP2010 Plus was used. The column temperature was programmed to rise to 200 ° C. at 5 ° C./min for 1 minute at an initial temperature of 150 ° C., then to 240 ° C. at 10 ° C./min and maintained for 5 minutes. Mass spectra were acquired and processed with GCMSsolution software (Shimadzu, Version 2.61). Free fatty acids and FAME standards were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Analysis of Lipid Species by LC-MS Mature individual single seeds were subjected to analysis of lipid total metabolites (LC) using LC-MS at School of Botany, University of Melbourne. Total lipids were extracted and dissolved in CHCl ₃ as described by Bligh and Dyer (1959). An aliquot of 1 mg lipid was dried over N ₂ and dissolved in 1 mM butanol: methanol (1: 1 v / v) in 10 mM butylated hydroxytoluene, Agilent 1200 Series LC and 6410B electrospray ionized triple quadruple. Analyzed using polar LC-MS. Lipids were chromatographed using an Ascentis Express RP-Amide column (5 cm x 2.1 mm, Supelco) and a binary gradient at a flow rate of 0.2 mL / min. Mobile _phase, A, _H 2 O: methanol: tetrahydrofuran (50: 20: 30, v / v / v) 10mM ammonium formate in; B. 10 mM ammonium formate in H ₂ O: methanol: tetrahydrofuran (5:20:75, v / v / v). Fatty acids 16: 0, 16: 1 18: 0, 18: 1, 18: 2, 18: 3 were used to select selected neutral lipids (TAG and DAG) and phosphocholine (PC) with a collision energy of 25V and Analyzed by multiple reaction monitoring (MRM) using a 135 V fragmentor. Individual MRM TAGs and DAGs were identified based on ammonia-treated precursor ions and product ions from neutral loss of fatty acids. TAG and DAG were quantified using a 10 uM Tristearin external standard.

Analysis of sterol content of oil sample About 10 mg of oil sample with a further aliquot of C24: 0 monol as an internal standard, 4 mL of 5% KOH in 80% MeOH, and teflon lined screw caps were attached. Saponification was carried out by heating at 80 ° C. for 2 hours in a glass tube. After cooling the reaction mixture, sterols were extracted into 2 mL of hexane: dichloromethane (4: 1 v / v) by adding 2 mL of Milli-Q water, shaking and vortexing. The mixture was centrifuged to remove the sterol extract and washed with 2 mL Milli-Q water. The sterol extract was then removed after shaking and centrifugation. The extract was evaporated using nitrogen gas vapor and the sterol was silylated using 200 mL BSTFA and heated at 80 ° C. for 2 hours.

For GC / GC-MS analysis of sterols, sterol-OTMSi derivatives were dried on a heat block at 40 ° C. under nitrogen gas vapor, then chloroform or GC / GC-MS analysis just prior to GC / GC-MS analysis. Redissolved in hexane. Agilent Technologies Agilent Technologies C 68A with a Superco Equity ™ -1 fused silica capillary column (15 m × 0.1 mm or 0.1 μm film thickness), FID, split / splitless injector and Agilent Technologies 7683B Series autosampler and injector The sterol-OTMS derivatives were analyzed by gas chromatography (GC) using Alto, California, USA. Helium was the carrier gas. Samples were injected in splitless mode at an oven temperature of 120 ° C. After the injection, the oven temperature was increased to 270 ° C. at 10 ° C. min ⁻¹ and finally increased to 300 ° C. at 5 ° C. min ⁻¹ . Peaks were quantified using Agilent Technologies ChemStation software (Palo Alto, California, USA). The GC results were subjected to an error of ± 5% of the individual component areas.

  GC-mass spectroscopy (GC-MS) analysis was performed on Finnigan Thermoquest GCQ GC-MS and Finnigan Thermo Electron Corporation GC-MS, both systems were equipped with on-column injector and Thermoquest Xcalib AxAUS Each GC was equipped with a capillary column having the same polarity as described above. Using mass spectral data, individual components were identified by comparing retention time data with those obtained against authentic and laboratory standards. Simultaneously with the sample batch, a blank analysis of the entire procedure was performed.

Quantification of TAG with Iatroscan 1 μL of each plant extract was loaded into one Chromarod-SII for TLC-FID Iatroscan ™ (Mitsubishi Chemical Media Corporation-Japan). The Chromarod rack is then placed in an equilibrated developer tank containing 70 ml of hexane / CHCl ₃ / 2-propanol / formic acid (85 / 10.716 / 0.567 / 0.0567 v / v / v / v) solvent system. Moved. After a 30 minute incubation, the Chromarod rack was dried at 100 ° C. for 3 minutes and immediately scanned on an Iatroscan MK-6s TLC-FID analyzer (Mitsubishi Chemical Media Corporation-Japan). The DAGE internal standard and the peak area of TAG are integrated using SIC-480II integration software (Version: 7.0-ESIC System Instruments Co., LTD-Japan).

  In two steps, TAG is quantified. First, the DAGE internal standard is scanned in all samples to correct the extraction yield, and then the concentrated TAG sample is selected and diluted. The amount of TAG is then quantified in the diluted sample using a second scan with external calibration using glyceryl trilinoleate (Sigma-Aldrich) as an external standard.

Expression of Candidate FAD2 Gene in Saccharomyces cerevisiae A DNA fragment containing the entire open reading frame of the candidate FAD2 cDNA is excised from the pGEMT-easy vector as an EcoRI fragment and placed in the corresponding site of the vector pENTR11 (Invitrogen, Carlsbad, CA, USA) Inserted. The insert was then cloned into the target expression vector pYES2-DEST52 using Gateway® Cloning recombination technology (Stratagene, LaJolla, CA, USA) for inducible gene expression in yeast cells. Placed in an open reading frame under the control of the GAL1 promoter. The gene sequence in the resulting plasmid was confirmed by DNA sequencing. The resulting plasmid and the pYES2-DEST52 vector lacking any cDNA insert as a control was introduced into cells of Saccharomyces cerevisiae strain YPH499 by lithium acetate-mediated transformation. Expression of these candidate FAD2 genes in yeast cells with or without the supply of exogenous fatty acid substrates is essentially as described in Zhou et al. (2006) as described above. Each experiment was performed in triplicate.

Gene expression genes in plant cells in the transient expression system system are described in Voinnet et al. (2003) and Wood et al. (2009) was expressed in Nicotiana benthamiana leaf cells using a transient expression system. A vector for constitutive expression of viral silencing suppressor expression P19 under control of the CaMV 35S promoter was obtained from the laboratory of Peter Waterhouse, CSIRO Plant Industry, Canberra, Australia. The chimeric binary vector 35S: P19 was introduced into Agrobacterium umefaciens strain AGL1. Also, all other binary vectors containing a promoter, often a coding region expressed in plant cells from the 35S promoter, were introduced into Agrobacterium umefaciens strain AGL1. Recombinant cells were treated with a selectable marker gene in a binary vector at 28 ° C. in 5 mL LB medium supplemented with 50 mg / L rifampicin and either 50 mg / L kanamycin or 80 mg / L spectinomycin. The plant was grown to a stationary phase. By centrifugation at 3000 xg for 5 minutes at room temperature before resuspension in 1.0 ml invasion buffer containing 5 mM MES, pH 5.7, 5 mM MgSO ₄ , and 100 μM acetosyringone. Bacteria from each medium were pelleted. The resuspended cell medium was then incubated at 28 ° C. with shaking for an additional 3 hours. A 10-fold dilution of each medium in infiltration buffer was then mixed with an equal volume of 35S: P19 medium, diluted in the same manner, and the mixture was fully swollen. It was infiltrated into the back side of the bentamiana leaf. Unless otherwise specified, the mixed media containing the gene to be expressed contained the 35S: P19 construct in Agrobacterium. The control infiltrate contained only the 35S: P19 construct in Agrobacterium.

  The Agrobacterium cell mixture was used to infiltrate the leaves and plants were generally grown for another 5 days after infiltration before harvesting leaf discs for total lipid isolation and fatty acid analysis. N. Ventamiana plants were grown in a growth cabinet at a constant 24 ° C. using an Osram 'Soft White' fluorescent lamp placed directly on a plant having a light intensity of about 200 lux with a light / dark cycle of 14/10 hours. Grown on. In general, 6-week-old plants were used for experiments and infiltrated almost fully expanded real leaves. To avoid slight differences, all non-infiltrated leaves were removed after infiltration.

Real-time quantitative PCR (RT-qPCR)
Gene expression analysis was performed by quantitative RT-PCR using a BIORAD CFX96 ™ real-time PCR detection system and iQTM SYBR® Green Supermix (BioRad, Hercules, CA, USA). Primers designed for gene-specific amplification that are 19-23 nucleotides long and have a melting temperature (Tm) of about 65 ° C., resulting in an amplification product of about 100-200 bp. PCR reactions were performed in 96 well plates. All RT-PCR reactions were performed in triplicate. The reaction mixture contained 1 × iQ ™ SYBR® Green Supermix (BioRad, Hercules, Calif., USA), 5 μM forward and reverse primers, and 400 ng cDNA and was used in a volume of 10 μL per well. The thermal cycle conditions were 95 ° C. for 3 minutes, followed by 40 cycles of 95 ° C. for 10 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 30 seconds. After the final step of PCR from 60 ° C. to 95 ° C. at 0.1 ° C./s, the specificity of PCR amplification was monitored by melting curve analysis. In addition, the PCR product was also checked for purity by agarose gel electrophoresis and confirmed by sequencing. The structurally expressed gene KASII was used as an endogenous reference to normalize expression levels. Data were calibrated to the corresponding gene expression level, followed by the 2- ^ΔΔCt method for relative quantification (Schmittgen, 2008). This data was presented as the mean ± standard deviation of three reactions performed on independent 96-well plates.

DNA Isolation and Southern Blot Analysis Safflower Seedling Genomic DNA, genotype “SU”, was obtained from Paterson et al. (1993) isolated from fully expanded leaves. Further purification was performed using a CsCl gradient as previously described (Liu et al., 1999). Ten μg aliquots of safflower genomic DNA were digested with eight different restriction enzymes: AccI, BglII, BamHI, EcoRI, EcoRV, HindIII, XbaI, and XhoI. Genomic DNA digested with each restriction enzyme was electrophoresed on a 1% agarose gel. Gels were soaked in 0.5M NaOH, 1.5M NaCl for 30 minutes and DNA was blotted onto Hybond-N ⁺ nylon membrane (Amersham, UK). The filters were probed with α-P ³² dCTP-labeled DNA fragments corresponding to the entire coding region of the safflower CtFAD2-6 gene as representative of the CtFAD2 gene family under low stringency hybridization conditions. Hybridization was performed overnight at 65 ° C. in a solution containing 6 × SSPE, 10% Denhardt's solution, 0.5% SDS, and 100 μg / mL denatured salmon sperm DNA. After hybridization and a brief wash in 2 × SSC / 0.1% SDS at 50 ° C., before autoradiography, the filters were washed 20 × 50 ° C. each time in 0.2 × SSC / 0.1% SDS. Washed 3 times per minute.

Transformation of safflower and Arabidopsis thaliana A chimeric vector containing the genes used to transform Arabidopsis The cells from the medium of transformed Agrobacterium introduced into the tumefaciens strain AGL1 were transformed using the floral dip method (Clowh and Bent, 1998) for transformation. Used to treat Sariana (ecological Columbia) plants. Using transformed Agrobacterium medium, Belide et al. (2011) introduced transformed safflower plants.

Example 2 Isolation of safflower cDNA, a candidate for encoding FAD2.
Total RNA Extraction and cDNA Synthesis To generate cDNA from safflower, total RNA was isolated from 100 mg frozen safflower tissue samples, including developing embryos, leaves, roots, and hypocotyls. This was done for each tissue separately using the RNeasy® Plant total RNA kit (Qiagen, Hilden, Germany) according to the supplier's protocol. The RNA concentration in the preparation was determined using a NanoDrop ™ spectrophotometer ND1000 (Thermo Fisher Scientific, Victoria, Australia) and the RNA concentration was made uniform before analysis. The quality and relative amount of RNA in each preparation was visualized by gel electrophoresis of the samples through a 1% (w / v) agarose gel containing formaldehyde. The RNA preparation was treated with RQ1 RNase-free DNase (Qiagen, Hilden, Germany) to remove contaminating genomic DNA. First-strand cDNA was prepared according to the manufacturer's instructions using the SuperScript III First-Strand Synthesis System (Qiagen, Hilden, Germany) using oligo (dT) ₂₀ primer and 400 ng of each DNA-restriction. Synthesized from the preparation.

Isolation of FAD2 cDNA expressing seeds from a developing seed cDNA library First , a cDNA library derived from a developing embryo of safflower genotype “SU” (wild type, high linoleic acid level) is screened Thus, safflower FAD2 cDNA expressing seeds was obtained. Library construction began with RNA extraction from a mixture of immature embryos of different developmental stages, harvested, ground and powdered in liquid nitrogen, and followed by TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. , USA) was used for RNA extraction. RNA containing poly (A) was isolated using a Qiagen mRNA purification kit (Qiagen, Hilden, Germany).

  First strand oligo (dT) primer cDNA was synthesized and converted to double stranded DNA using the Stratagene cDNA synthesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Blunt end cDNA was ligated with an EcoRI adapter, phosphorylated, and size fractionated by gel filtration in a Chromaspin + TE-400 column (Clontech, CA, USA). Recombinant cDNA was propagated in E. coli strain XL-1 Blue MRF 'using the Stratagene Predigitated Lambda ZAP II / EcoRI / CIAP cloning kit.

To identify FAD2 clones, a DNA fragment corresponding to the coding region of Arabidopsis FAD2 (GenBank Accession No. L26296) was used according to a previously described protocol (Liu et al., 1999) The rally was screened. Positive plaques were purified through two successive rounds of screening, and purified phagemids containing putative FAD2 cDNA were excised as outlined in the instructions for the Stratagene λZAPII cDNA synthesis kit. Sequence analysis of the FAD2 sequence was performed by the NCBI Blast program ( www.ncbi.nlm.nih.gov/BLAST/ ). An open reading frame was predicted by using VectorNTI. Two different full-length cDNAs were isolated from the growing seed cDNA library and designated CtFAD2-1 and CtFAD2-2, respectively.

Identification of ESTs for Candidate FAD2 Genes To identify additional candidate FAD2 cDNAs, the expressed sequence tag (EST) database (cgpdb.ucdavis.edu/cgpdb2.) Of the safflower Composite Genome Project (CGP) It was probed using the program BLASTp for ESTs that encoded a polypeptide with similarity to thaliana FAD2 (GenBank accession number L26296). At least eleven distinct FAD2 cDNA sequence contigs were identified, of which two contigs showed identical sequences with CtFAD2-1 and CtFAD2-2 isolated from a safflower seed cDNA library. In addition, nine different cDNAs were identified and designated as CtFAD2-3 to CtFAD2-11, respectively.

3 'and 5' RACE
The longest EST clone of each of the 9 contigs (CtFAD2-3 to CtFAD2-11) was selected as the starting point for the isolation of the corresponding full-length cDNA sequence. The process is rapid amplification of 3′- and 5′- cDNA ends (RACE) using cDNA generated from RNA from various safflower tissues including developing embryos, leaves, roots, hypocotyls, and flowers. It was used. Gene specific primers (GSP) were designed from the longest EST clone of each contig. 3'-RACE was performed using a one-step RT-PCR kit (Bioline, London, UK) according to the manufacturer's instructions. A gene specific primer (GSP) was used in the first round of each PCR amplification of ESTs selected in combination with a poly (dT) primer at the NotI site at its 3 ′ end. A second round of PCR was performed on the product of the first round using GSP nested in combination with poly (dT) primers. The GSP for 3'RACE is listed in Table 2.

  The 5 'end of the CtFAD2-6 cDNA was cloned using a 5'RACE System kit (Invitrogen, Carlsbad, CA, USA). Only the CtFAD2-6 mRNA was reverse transcribed into cDNA using the gene specific primer GSP1, 5'-ACCTAACGACAGTCATGAACAAG-3 '(SEQ ID NO: 76). Nested gene specific primer GSP2, 5'-GTGAGGGAAAGCGGAGTGGACAAC-3 '(SEQ ID NO: 77) was used during the first PCR amplification. The reaction conditions were 33 cycles of hot start at 95 ° C. for 4 minutes, denaturation at 94 ° C. for 45 seconds, annealing at 55 ° C. for 1 minute, and extension at 72 ° C. for 2 minutes before adding the polymerase. .

  Amplified 3 'and 5' fragments were subcloned into the vector pGEM-Teasy and sequenced from both directions. Sequence comparison of the 3 ′ and 5 ′ end cloned fragments with the corresponding ESTs showed overlapping regions that were consistent with each other, thereby providing 3 ′ and 5 ′ sequences for each gene, and 11 cDNAs Allows assembly of putative full-length sequences for each of the

Isolation of Full-Length cDNA Sequences for Candidate CtFAD2 Genes Several safflower tissues including developing embryos, leaves, roots, hypocotyls, and flowers to isolate the coding region of full-length proteins for nine CtFAD2 genes Total RNA from was used to amplify the ORF using a one-step RT-PCR kit (Stratagene, La Jolla, CA, USA). The primers used to amplify the ORF (Table 3) were based on DNA sequences located in the 5 'and 3' UTRs of each cDNA. Amplified PCR products were cloned into the vector pGEM-Teasy® and their nucleotide sequences were obtained by DNA sequencing.

Candidate FAD2 sequence features from safflower Feature 11 cDNA features are summarized in Table 4 and polypeptide features are summarized in Table 5.

The predicted amino acid sequences of the encoded polypeptides CtFAD2-1 to CtFAD2-11 shared a wide range of sequence identity with about 44% to 86% identity to each other. They showed 53% to 62% sequence identity with Arabidopsis FAD2. The predicted polypeptide size was in the range of 372-388 amino acids. That is, they were all about 380 amino acid residues long. The cDNA has unique 5 ′ and 3 ′ untranslated region (UTR) sequences, so endogenous genes could be easily recognized by their UTR sequences. Amplification of protein coding regions from safflower genomic DNA resulted in identical DNA sequences with corresponding cDNAs for each of the 11 genes, indicating that there were no introns that interrupted those protein coding regions. It was.

  To examine the relationship of safflower candidate FAD2 polypeptides to known FAD2 enzymes, 11 putative polypeptide sequences were aligned with plant FAD2 sequences and a proximity-binding tree was constructed using the vector NTI (FIG. 1). . As shown in FIG. 1, the amino acid sequences of CtFAD2-1 and CtFAD2-10 were most closely related to each other and then to FAD2 that expressed seeds from other species first. CtFAD2-2 was more closely related to structurally expressed genes from other species in safflower than other candidate FAD2. CtFAD2-3, -4, -5, -6, and -7 are most likely as a result of the evolution of recently branched genes that form new branches in the proximal junction tree and multiply in safflower. Interestingly, in terms of syngeneicity to other species, these were most closely related to functionally branched FAD2 conjugase from Calendula officinalis. FAD2-11 was more closely related to acetylenases from several plant species, including sunflower vFAD2 induced by a fungal inducer (Cahoon et al., 2003). CtFAD2-8 and -9 appeared to branch further than other candidate FAD2 from safflower. However, this analysis also gave some clues about the functions that can be sequence-compared, but by itself it also showed that it could not provide reliable conclusions about the functions of different FAD2 candidates. Therefore, functional analysis was required to draw conclusions about the function of each gene / polypeptide.

Sequence comparison showed that safflower candidate FAD2 polypeptides share about 50% -60% sequence identity and 52% -65% similarity with known FAD2 enzymes from other species. The extent of DNA sequence dispersion among safflower CtFAD2 genes is that CtFAD2-3, -4, and -5 are all more similar to each other than CtFAD2-1 or CtFAD2-10, and vice versa Are reflected in their phylogenetic relationships. . These numbers have close agreement in the amino acid identity matrix (Table 6).

Candidate CtFAD2 Polypeptide Feature 11 Each predicted polypeptide of candidate CtFAD2 contained an aromatic amino acid rich motif at the C-terminal extreme. Such motifs have been identified in other plant FAD2 polypeptides and are thought to be necessary to maintain localization in the ER (McCartney et al., 2004). Consistent with other plant membrane bound fatty acid desaturase enzymes, each predicted CtFAD2 polypeptide contained three histidine-rich motifs (His boxes). Such His-rich motifs are highly conserved in the FAD2 enzyme and have been shown to be involved in the formation of ferric oxide complexes used in biochemical catalysis (Shanklin et al., 1998). In most candidate CtFAD2 polypeptide sequences, the first histidine motif was HECGHH, with the exception of CtFAD2-5 and -6 with HDCGGHH and HDLGHH, respectively. The last amino acid in the first His box (HECGHQ) in CtFAD2-8 was Q rather than H. We searched for this motif in 55 known plant FAD2 enzymes, and the conversion from H to Q is also present in branched FAD2 homologues derived from Lesquerella lindheimeri, which mainly has fatty acid hydroxylase activity ( Genbank accession number EF432246, Dauk et al., 2007). The second histidine motif was highly conserved as the amino acid sequence HRRHH in several candidate safflower FAD2, including CtFAD2-1, -2, -8, -9, and -10. The amino acid N is found in CtFAD2-11 at position +3 of the motif, Crepis alpina CREP1, Crepis paraestina Cpal2, and sunflower vFAD2 (AY166773.1), Calendula officinalis It was noteworthy that it was also found in a number of functionally branched FAD type 2 enzymes, including FAC2 (AF33044.1), Rudbeckia hirta acetylenase (AY1667676.1). The amino acid at this position in CtFAD2-3, -4, -5, -6, and -7 was either S or T.

  In each of the CtFAD2-1, -2, -9, and -10 polypeptides, the amino acid directly preceding the first histidine box was alanine, which is the same as for other plant fatty acid Δ12-desaturase enzymes. The amino acid valine (V) but not alanine was present at that position in CtFAD2-5, while the other six CtFAD2 polypeptides had glycine at this position. It has been found in Cahoon et al. That a glycine substitution of alanine at this position was found in functionally branched FAD2 enzymes except for fatty acid Δ12-hydroxylase. (2003). As described in the Examples below, subsequent heterologous expression experiments testing candidate functions indicated that each of the CtFAD2-1, -2, and -10 polypeptides was an oleate Δ12-desaturase. However, CtFAD2-9 showed desaturase specificity to palmitate (C16: 1) but not oleate.

  Of the 11 candidate CtFAD2, only the CtFAD2-11 polypeptide suggests that it occurs in all plant acetylenases (D / N) from the −5 of the third histidine box consistent with the VX (H / N) motif Means having a DVTH sequence at position 2 (Blacklock et al., 2010). For other known plant FAD2 oleate desaturases, the five amino acids immediately following the third histidine box of CtFAD2-1, -2, and -10 polypeptides were LFSTM. In contrast, the palmitate-specific fatty acid desaturase CtFAD2-9 had an LFSYI motif at this position with two amino acid substitutions at positions +4 and +5. In CtFAD2-3, -4, and -5 polypeptides, S is from position +3, derived from Calendula officinalis (FAC2, Accession AAK26632) and Trichosanthes kirilovii (Accession AAO37751). Substituted with P present only in other FAD2 fatty acid conjugases including.

  Serine-185 of soybean FAD2-1 enzyme has been shown to be phosphorylated during seed development as a regulatory mechanism for its enzyme activity (Tang et al., 2005). Of the 11 candidate CtFAD2 polypeptides, only CtFAD2-1 had serine (serine-181) at the corresponding position relative to soybean FAD2-1. Conclusion It is concluded that the same post-translational regulatory mechanism operates during safflower seed development and oil accumulation through phosphorylation of serine-185 to regulate desaturation of microsomal Δ12 oleate in developing seeds It was attached.

Example 3 Isolation of genomic sequences for FAD2 candidates
Isolation of the 5′UTR intron of the candidate CtFAD2 gene The intron-exon structure of the FAD2 gene is conserved in many flowering plants. All the FAD2 genes studied so far are located in the 5'UTR, with one exception that the intron of soybean FAD2-1 is located in the coding region immediately after the first ATG translation initiation codon. Only one intron (Liu et al., 2001, Kim et al., 2006, Mroczka et al., 2010). Intron sequence branching can be used as a measure of evolution between taxonomically related species (Liu et al., 2001).

  To isolate the DNA sequence of possible introns located within the 5′-UTR of the candidate CtFAD2 gene, a typical intron splice site (AG: GT) is predicted in the 5′UTR of the respective CtFAD2 cDNA sequence. PCR primers were designed based on the predicted splice site flanking sequences. These primers are listed in Table 7. Genomic DNA isolated from safflower genotype SU was used as a template for the PCR reaction to amplify the genomic region corresponding to the 5 'UTR. Amplification was achieved in a 50 μL reaction containing 100 ng genomic DNA, 20 pmol of each primer, and Hotstar supplied by the manufacturer (Qiagen, Hilden, Germany). The PCR temperature cycle was 1 cycle at 94 ° C for 15 minutes, 94 ° C for 30 seconds, 55 ° C for 1 minute, and 72 ° C for 2 minutes, using Kyratec supercycler SC200 (Kyratec, Queensland, Australia) Performed at 72 ° C. for 10 minutes. The PCR product was cloned into pGEM-T Easy and then sequenced.

We have predicted from 8 of 11 candidate CtFAD2 genes, designated CtFAD2-1, -2, -3, -4, -5, -7, -10, and -11. 5 'intron could be obtained. The main features of these introns are shown in Table 8. The intron was not successfully amplified from CtFAD2-6, -8, and -9, presumably due to the insufficient length of the 5'UTR in which the intron was present. In higher plants, intron deletions from nuclear genes are usually observed, but FAD2 with low introns was not reported (Loguercio et al., 1998, Small et al., 2000a; b).

  Each intron sequence of each of the eight genes is located within the 5′-UTR of each gene at a position spanning 11-38 bp upstream of the putative translation start codon of the first ATG in each open reading frame. Located. The intron length ranged from 104 bp (CtFAD2-11) to 3,090 bp (CtFAD2-2) (Table 8). For CtFAD2-1, intron sizes are from Arabidopsis Information Resource, http://www.arabidopsis.org, cottonseed (Liu et al., 2001), and sesame indicum from Sedum indicum. It was 1,144 bp of the same size as the intron identified in the gene (Kim et al., 2006). The putative splice site dinucleotides AG and GT were conserved in all eight of the examined CtFAD2 genes, otherwise all intron sequences had any significant homology between them. Branched in no sequence. All intron sequences were A / T rich with an A / T content between 61% and 75%, consistent with many other intron sequences from dicotyledonous plants. In other dicotyledonous genes, the Arabidopsis FAD2 gene had a 1,134 bp intron that was only 5 bp upstream from its ATG translation initiation codon. The size of the 5'-UTR intron of the Gossypium hirstum FAD2-1 gene was 1,133 bp, and was located 9 bp upstream from the translation start codon. In contrast, cottonseed FAD2-4 and FAD2-3 genes had larger 5'-UTR introns of 2,780 bp and 2,967 bp, respectively, and were located 12 bp upstream from the translation initiation codon. Each candidate CtFAD2 gene can be distinguished by the location and size difference of the 5'-UTR intron in each gene. This difference can also be important in providing differential expression of genes. Such introns have been reported to have favorable effects on reporter gene expression in sesame (Kim et al., 2006). The corresponding intron has been shown to be an effective target for post-translational gene silencing of FAD2 in soybean (Mroczka et al., 2010).

  It is known that introns can have dramatic effects on gene expression profiles. Analysis of intron sequences by the PLACE program (www.dna.affrc.go.jp/PLACE/) identified several putative cis-regulatory elements. For example, motifs such as ABRE and SEF4 that are normally present in seed-specific promoters are located in CtFAD2-1. The AG motif normally found in defense-related gene promoters induced by various stresses such as injury or treatment with inducers is located in CtFAD2-3 that is specifically expressed in safflower young seedling hypocotyls and dicotyledons did.

Example 4 Southern blot hybridization analysis of candidate safflower FAD2 gene In safflower, the complexity of the FAD2-like gene family was examined by Southern blot hybridization analysis. Low stringency hybridization analysis indicated that FAD2 was encoded by a complex multigene family in safflower (FIG. 2). By counting hybridizing fragments obtained by using various restriction enzymes that cleave genomic DNA, it was speculated that there were more than 10 FAD2 or FAD2-like genes in safflower. The difference seen in the intensity of hybridization for different fragments is probably correlated with the relative level of sequence identity and the probe DNA used. Safflower is a diploid species and a single wild progeny species C. It is thought to have paraestinus (Chapman and Burke, 2007). We speculate that the unusually large FAD2 gene family in safflower is probably derived from replication of several ancient genes, resulting in the specialization and specific activity of different members of the gene family.

Embodiment 5 FIG. Functional analysis of candidate genes in yeast and plant cells
Expression of Candidate CtFAD2 Gene in Yeast-Functional Analysis To study the functional expression of several plant FAD2 Δ12 oleate fatty acid desaturases as convenient host cells, yeast S. cerevisiae has been used (Covello and Reed 1996, Dyer et al., 2002, Hernandez et al., 2005). S. Cerevisiae has a relatively simple fatty acid profile and contains sufficient oleic acid within its phospholipid that can be used as a substrate for the FAD2 enzyme. It also lacks endogenous FAD2 activity. Therefore, as described in Example 1, eleven candidate CtFAD2 genes were tested in yeast strain YPH499 using the respective open reading frame under the control of the pYES2-derived construct, GAL1 promoter.

As shown in FIG. 3, when analyzing the fatty acid composition of yeast cells containing the “empty vector” pYES2, the linoleic acid (18: 2) was predicted as expected because the yeast lacks endogenous FAD2. Neither hexadecadienoic acid (16: 2) was detected. In contrast, the gas chromatograms of fatty acids obtained from yeast cells expressing CtFAD2-1, CtFAD2-2, and CtFAD2-10 open reading frames are each 11.293 minutes corresponding to linoleic acid (C18: 2). The gas chromatograms for CtFAD2-9 and CtFAD2-10 showed a fatty acid peak with a retention time of 8.513 minutes corresponding to C16: 2. These data indicated that CtFAD2-1, CtFAD2-2, and CtFAD2-10 were able to convert oleic acid to linoleic acid and therefore were Δ12 oleate desaturases. However, the level of 18: 2 produced was lower than that for the Arabidopsis AtFAD2 construct used as a positive control. CtFAD2-10 uses both linoleic acid (C18: 2) and hexadecadienoic acid (C16: 2) using oleic acid (C18: 1) and palmitoleic acid (C16: 1) as substrates, respectively. Although produced, CtFAD2-9 desaturates palmitoleic acid and was therefore a Δ12 palmitoleate desaturase. Two new micropeaks appearing in the chromatogram of FAME derived from yeast cells expressing CtFAD2-11 were linoleic acid (18: 2 ^{Δ9 (Z), 12 ()} by their pyrrolizide adduct and DMOX GC-MS. ^Z) ) and its trans isomer (18: 2 ^{Δ9 (Z), 12 (E)} ) (FIG. 3H). Table 9 summarizes the fatty acid composition of yeast cells expressing the CtFAD2 coding region. No new peaks were detected in yeast cells expressing CtFAD2-3, -4, -5, -6, -7, and -8.

  To test whether any of the candidate CtFAD2 polypeptides have fatty acid hydroxylase activity, FAMEs prepared from yeast cells expressing each of the CtFAD2 open reading frames were converted to TMS- hydroxyl residues. The mass spectrum could be tested by reacting with a silylating reagent that converts to an ether derivative. However, hydrixyl derivatives of common fatty acids such as oleic acid were not detected in any of the yeast cell lines expressing the candidate CtFAD2 open reading frame. This indicates that none of the 11 CtFAD2 genes encoded a polypeptide having fatty acid hydroxylase activity in yeast.

Activity of Δ12-epoxygenase and Δ12-acetylenase (both using linoleic acid as a fatty acid substrate) by supplementing the growth medium of the same yeast cell line with free linoleic acid and then analyzing the fatty acid composition Further experiments were performed to detect. In order to express the construct, supplementation was performed after adding galactose to the medium. In the gas chromatogram, no new fatty acid peaks were detected, including those representing epoxy and acetylenic fatty acid derivatives. Heterologous expression of these novel fatty acids in yeast with supplementation of exogenous free fatty acids has encountered several difficulties in showing activity (Lee et al., 1998, Cahoon et al., 2003). Therefore, functional analysis in plant cells was performed as follows.

N. In order to express the gene in a structural manner in plant leaves, in particular in CtFAD2 ORF, an enhanced CaMV-35S promoter and a polyadenylation signal are expressed in a transient expression plant cell in a candidate CtFAD2 gene in Benthamiana. It was inserted in sense orientation into a modified pORE04 binary vector between the nos 3 ′ terminator containing the sequence (Coutu et al., 2007) (SEQ ID NO: 54). Previous studies have shown that transgene expression is significantly enhanced by co-expression of the viral silencing suppressor protein P19; It was shown that in a transient assay based on Benthamiana leaves, host transgene silencing was induced (Voynet et al., 2003, Wood et al., 2009, Petrie et al., 2010). These experiments were performed as described in Example 1.

As described above, the function of CtFAD2-11 is first described in S.F. As assessed by expression in S. cerevisiae, two new fatty acids were identified by GC-MS as 18: 2 ^{Δ9 (Z), 12 (Z)} and 18: 2 ^{Δ9 (Z), 12 (E)} , respectively. Consistent with the results obtained from yeast, Expression of CtFAD2-11 in Benamiana leaves gave a novel 18: 2 trans isomer. The methyl ester of this isomer showed a GC retention time identical to that of methyl 18: 2 ^{Δ9 (Z), 12 (E)} (FIG. 4B). The new 18: 2 ^{Δ9 (Z), 12 (E)} accounted for 0.35% of the fatty acids in the leaves after transient expression of CtFAD2-11 (Table 10). In addition, another new peak that was not observed in the yeast medium was detected. The total ion chromatogram and mass spectrum of this new fatty acid is consistent with that of crepenic acid (18: 2 ^{Δ9 (Z), 12 (c)} ) (FIGS. 4B and C), indicating that the CtFAD2-11 polypeptide is It shows having Δ12-acetylenase activity. As shown in Table 10, crepenic acid accounted for 0.51% of total fatty acids.

N. It was observed that transient expression of CtFAD2-11 in Benamiana cells resulted in a decrease in the content of 18: 2 ^{Δ9 (Z), 12 (Z)} compared to untransformed controls (Table 10). This is due to N. cerevisiae in the available pool of oleic acid substrates for both enzymes. It was more likely due to competition between CtFAD2-11 and endogenous cis-Δ12 oleate desaturase in Benamiana cells. In general, yeast and N. coli. The results of the Benamiana expression experiment showed that CtFAD2-11 primarily functioned as an oleate Δ12-desaturase lacking stereospecificity resulting in both linoleic acid and its trans-Δ12 isomer. Furthermore, the linoleic acid with a Δ12 double bond could be further unsaturated to form an acetylenic bond of crepenic acid.

In addition, the other 10 candidate CtFAD2 polypeptides were tested in the same manner as N.F. N. cerevisiae that was transiently expressed in Benamiana leaves but already has high levels of FAD2. We did not observe any new fatty acids that were not endogenously present in the Benamiana leaves.

Discussion The eleven candidate CtFAD2 genes identified in safflower represent the largest FAD2 gene family observed in any plant species tested so far. Although only a single FAD2 gene has been identified in Arabidopsis (Okuley et al., 1994), FAD2 appears to be encoded by a synonymous gene in most other plant genomes studied to date. Two distinct FAD2 genes are found in soybean (Heppard et al., 1996), flax (Fofana et al., 2004, Khadake et al., 2009), and olive (Hernanze et al., 2005), three in sunflower. Genes (Martinez-Rivas et al., 2001) and Camerina sativa (Kang et al., 2011), described in five genes in cottonseed (Liu et al., 1998). In the amphitetraploid species of brush kanapus, 4-6 different FAD2 genes have been identified in each diploid subgenome (Scheffler et al., 1997). All of the candidate CtFAD2 genes were expressed in safflower plants because these sequences were isolated from cDNA. This was further tested as described in Example 6.

  Although lacking comparable studies, it is clear that safflowers are unique with respect to the evolution of the FAD2 gene family. Safflower is a self-pollinating diploid plant species that is most closely related to the wild diploid species Carthamus paraestinus and is known to have extensive genome duplication or rearrangement. (Chapman and Burke, 2007). Because the candidate FAD2 gene did not contain an intron in the coding region sequence, multiple FAD2 cDNAs identified could not be attributed to alternative splicing. Rather, gene duplication was likely to be involved in generating the complexity of the FAD2 family in safflower. The phylogenetic tree topology showed that gene duplication can occur at several hierarchical levels. For example, CtFAD2-3, -4, and -5 polypeptides are more closely related to others in that there was a clade than other safflower FAD2 sequences, and more recent gene duplications of this clade Indicates that it can be involved in the appearance.

Example 6 Expression level of FAD2 candidate gene in safflower
Expression profile of FAD2 gene in different tissues RT-PCR analysis as described in Example 1 was performed to determine the tissue expression pattern of various candidate CtFAD2 genes. Total RNA is extracted from cotyledons, hypocotyls, roots, and leaf tissues from safflower seedlings of 10 DAG high linoleic acid genotype SU, and flower tissues from developing plants and developing embryos and used in these assays did. The oligonucleotide primers used for analysis are listed in Table 11.

  The temporal and spatial expression of 11 CtFAD2 genes is shown in FIG. RT-qPCR assay showed that only CtFAD2-1 was expressed in the growing seed. In contrast, CtFAD2-2 was expressed at low levels in seeds, like other tissues tested. Furthermore, CtFAD2-4, -5, -6, -7, -8, -9 was not observed in the developing embryo. Low but detectable levels of CtFAD2-10 and -11 expression were observed in the growing seed and also at the late developmental stage when safflower seeds reached maturity. CtFAD2-4, -6, -7, -9, and -11 all showed high levels of expression in young seedling tissues including cotyledons and hypocotyls. CtFAD2-5 and -8 and CtFAD2-10, which appeared to be root specific, are selectively expressed in floral tissues and contain relatively low levels of developing seeds and 10-day-old seedling tissues Detected in various other tissues tested.

  In a control reaction using total RNA template but no reverse transcriptase, no amplification product was detected after 40 cycles of amplification, indicating the absence of contaminating genomic DNA in the RNA preparation.

Example 7 Demonstration of a genetic mutant in safflower strain S317 The first identified high oleic acid characteristics in safflower found in safflower introduction from India are partially recessive, denoted ol at a single locus OL Dominated by alleles (Knowles and Hill, 1964). The oleic acid content of the olol genotype was typically 71-75% in greenhouse-grown plants (Knowles, 1989). Knowles (1968) incorporated the ol allele into the safflower breeding program and in the United States released the first high oleic acid (HO) safflower variant “UC-1” in 1966, followed by improved variants Saffola series including “Oleic Leed” and Saffola 317 (S-317), S-517, and S-518 were released. The high oleic acid (olol) genotype was relatively stable at different temperatures (Bartholomew, 1971). In addition, Knowles (1972) also described different alleles ol ₁ at the same locus generated with 35-50% oleic acid homozygous conditions. In contrast to olol genotype, ol ₁ ol ₁ genotype showed a strong response to temperature (Knowles, 1972).

  Additional genetic resources with higher oleic acid content (greater than 85%) have been reported (Fernandez-Martinez et al., 1993, Bergman et al., 2006). The oleic acid content of up to 89% in safflower was originally reported in Fernandez-Martinez et al. In Genetic Resource Accession I401472 originating from Bangladesh. (1993). Bergman et al. The Montola series developed by (2006) is more than 80%, clearly exceeding the highest level of oleic acid, in the “UC-1” variant containing the olol allele described by Knowles and Hill (1964). Contains oleic acid. Genetic and segregation analysis by crossbreeding, high oleic acid and very high oleic acid strains suggested that these two strains share the same allele at the OL locus. Very high oleic acid content (85%) was obtained by combining the ol allele and modifying a gene with a small positive result for oleic acid (Hamdan et al., 2009).

In Vitro Biochemical Characterization of High Oleic Acid Mutant S-317 As described by Stymne and Appelqvist (1978), safflower microsomes become highly olein at the middle maturation stage about 15 days after flowering (DPA). Freshly prepared from developing seeds of acid genotype S-317. A standard 90 μL reaction mixture contained 40 μg microsomal protein, 2 nmol [ ¹⁴ C] oleoyl-CoA in 0.1 mmol potassium phosphate buffer pH 7.2. 10 μL of 50 mM NADH was then added and the incubation continued for an additional 5, 10 or 20 minutes. The reaction was stopped by adding 90 μL 0.15 M acetic acid and the lipids were extracted with 500 μL CHCl ₃ : MeOH (1: 1). The lower CHCl ₃ phase is recovered and the polar lipids from it are thin filmed using a solvent system of CHCl ₃ / MeOH / HAc / H ₂ O (90: 15: 10: 3 v / v / v / v) Separation by chromatography (TLC). The spot corresponding to PC was scraped from the plate and the associated fatty acid groups were transmethylated in 2 ml of 2% sulfuric acid in MeOH at 90 ° C. for 30 minutes. The obtained FAME was separated on a TLC plate treated with AgNO ₃ with hexane: DEE: HAc (85: 15: 1 v / v / v). ¹⁴ C-labeled oleate and linoleic acid methyl ester standard were spotted on the plate as a reference. Plates were exposed and analyzed by Fujifilm FLA-5000 phosphorimager. The radiation of each sample was quantified with Fujifilm Multi Gauge software.

Upon addition of NADH to the reaction by wild type microsomes, it was observed that the added [ ¹⁴ C] oleoyl-CoA disappeared immediately within 10 minutes upon the appearance of [ ¹⁴ C] linoleate, This indicated an efficient conversion of oleate to linoleate in wild-type safflower microsomes. In contrast, for high oleic acid genotype S-317, a significantly higher ratio of [ ¹⁴ C] oleate to [ ¹⁴ C] linoleate was found in in vitro reactions over time (Table 12), We showed that the biosynthesis of linoleic acid by desaturation of oleate by microsomes was dramatically reduced in this genotype.

Molecular characterization of the high oleate allele ol To understand the molecular basis of the high oleic acid genotype (olol) in safflower, FAD2 cDNA expressed by two seeds, namely CtFAD2-1 and CtFAD2-2, Amplified and sequenced by PCR from three high oleic acid variants: S-317, LeSaf 496, and CW99-OL. CDNAs covering the entire coding region of the CtFAD2-1 gene from all three high oleic acid variants are identified in each other in nucleotide sequence and share approximately 98% sequence identity with the CtFAD2-1 cDNA from the wild type variant SU This included one nucleotide deletion and 22 nucleotide substitutions in the HO genotype compared to the wild type. A single base pair deletion was found at nucleotide 606, counting from the first ATG approximately in the middle of the CtFAD2-1 coding region. This deletion results in a shift in the post-translational reading frame that created a stop codon immediately after the deletion, so that of the three olol variants encoding the predicted truncated polypeptide that did not contain a third histidine box. The mutated gene was present in the wild type protein (FIG. 6). It is worth noting that there was a relatively high level of sequence variation in the DNA sequence near the deleted single nucleotide site of the ol allele suggesting that additional mutations were accumulated in the mutated gene. did. .

  In addition, a DNA region containing the 5 'UTR intron of CtFAD2-1 and CtFAD2-2 was isolated from the olol mutation S-317 and compared to the wild type intron. The CtFAD2-1 intron from S-317 was 1144 bp in length and 61 bp longer than the 1083 bp long wild-type SU intron. Comparison of the nucleotide sequence of the CtFAD2-1 intron showed 76.8% overall sequence identity, indicating that this intron was different in that there were 27 disappearances and 95 single nucleotide substitutions (FIG. 7). ).

  Interestingly, nucleotide substitutions in the mutated gene are not evenly distributed in the 1142 bp long region corresponding to the coding region of the defective CtFAD2-1, with 14 out of 22 (63.6%) substitutions missing the nucleotide Nearly, most were within 123 bp just downstream of the single nucleotide deletion. In contrast, the CtFAD2-2 intron in the wild type and mutant genotypes shared 99.5% overall sequence identity with only 12 nucleotide substitutions and one 2-nt loss. This may be because some selection pressure occurred in the defective CtFAD2-1 gene in the HO mutant, or perhaps the CtFAD2-1 mutation was of an old origin, It shows that it is more likely to have origins in safflower progeny such as Paraestinus.

A commercially available EMS mutant (S-901) from high oleic acid variant S-518 is described in US Pat. No. 5,912,416. Genetic studies have shown that the so-called ol ₂ allele of this new genotype is different from the ol and ol ₁ alleles at the OL locus, but its molecular nature is determined by Weisker (US Pat. No. US 5,912). , 416). The S-901 genotype was characterized by increased levels of oleic acid relative to 89.5-91.5% total fatty acids in mature seeds. Reduction of saturated fatty acids, ie, about 4% reduction with palmitic acid and about 2.5% reduction with stearic acid. However, S-901 did not show a normal plant phenotype and struggled to include growth and yield. Morphologically, it was short compared to its parent strain S-518 and the flower head was small. It also flowered late and there was less oil in the seeds.

A single nucleotide deletion sequence polymorphism in the mutant CtFAD2-1 allele that is concluded to be the causative mutant involved in the complete PCR marker design HO phenotype for high oleic acid reproduction Developed as a molecular basis for highly efficient molecular markers to track mutant ol alleles. Therefore, we have developed a molecular marker assay that allows the identification and selection of mutant ol alleles for breeding purposes or variant specific purposes, even when it was present in a heterozygous state. . Thereby, molecular marker assisted selection eliminates the need to generate extra generations of plants that must be screened for the fatty acid phenotype. Simple genetics combined with a complete molecular marker assay will allow safflower breeders to quickly incorporate high oleic acid attributes into their breeding programs.

  In order to easily generate differential markers based on PCR reactions, the exons of CtFAD2-1 between wild-type SU and high oleic acid genotype S-317 had insufficient sequence mutations. It seemed like it was. However, we have successfully exploited the relatively high sequence variance of the 5'UTR intron of CtFAD2-1 between the OL and ol alleles. There was a highly variable sequence extension between these two alleles that allowed the design of unique PCR primers. The following exemplary primers were designed to amplify a specific product 315 bp long from a high oleic acid genotype carrying an olol mutant allele but not wild type SU. HO-sense: 5'-ATAAGGCTGGTTCACGGGTTT-3 '(SEQ ID NO: 140) and HO-antisense: 5'-GCTCAGTTGGGGATACAAGGAT-3' (SEQ ID NO: 141) (Figure 7). Another pair of exemplary primers specific for the wild type gene in the variant SU resulted in a 603 bp PCR product as follows: HL-sense: 5′-AGTTATGGTTCGATGATCGACG-3 ′ (SEQ ID NO: 142) And HL-antisense: 5'-TTGCTATACATATTGAAGGCACT-3 '(SEQ ID NO: 143) (Figure 7). A pair of primers derived from the safflower KASII gene, ctkasII-sense: 5'-CTGAACTGCAATTATTTAGGG-3 '(SEQ ID NO: 144) and ctkasII-antisense 5'-GGTATTGGTATTGGATGGGGCG-3' (SEQ ID NO: 145) are equivalent to the template DNA. And was used as a positive control to establish good PCR performance.

  The PCR reaction conditions were 94 ° C. for 2 minutes, followed by 40 cycles of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 30 seconds. The reaction products were separated by electrophoresis on a 1% agarose gel and visualized under ultraviolet light after ethidium bromide staining of the gel. An approximately 300 bp fragment was observed for amplification reactions against all five high oleic acid genotypes tested, namely S-317, S-517, CW99-OL, LeSaf496, and Ciano-OL. The fragment was absent from wild type genotype SU. Conversely, an approximately 600 bp fragment was present in the amplification against wild type safflower SU, but not in any of the high oleic acid variants tested. As a positive control, a 198 bp band from the KASII gene was amplified with the reaction for all of the strains tested. The identity of the amplicon was confirmed by DNA sequencing.

  The sequence variance within the 5′UTR intron region of the CtFAD2-1 gene between the high oleic acid allele and the wild type safflower allele thereby causes the PCR marker diagnosis due to the presence or absence of the CtFAD2-1 mutation. Make development easier. It was fully linked to the ol allele whatever the genetic background, ie it was a fully linked marker. However, molecular markers are dominant markers, and as a result, the use of only that marker could not allow discrimination between homozygous and heterozygous genotypes in the ol allele. To overcome this, another pair of PCR primers was designed that only amplifies the wild type Ol allele. Therefore, the use of such wild type specific primers in combination with high oleic acid specific PCR primers allows to distinguish between homozygous and heterozygous genotypes at the CtFAD2-1 locus I made it.

CtFAD2-1 expression is dramatically reduced in high oleic acid genotypes.
In the upper section, CtFAD2-1 was expressed only in developing seeds and was not detected in various other tissues tested including leaves, roots, flowers, cotyledons and hypocotyls from young safflower seedlings showed that. CtFAD2-1 was highly expressed in developing seeds with a high rate of fatty acid metabolism, resulting in an active oil accumulation with mostly C18: 2 in a relatively short period of time. CtFAD2-1 had its highest expression level at an approximately midpoint during seed development and a more moderate expression level at both early and late stages of seed development.

  Using the RT-qPCR assay method, the level of normalized gene expression of the CtFAD2-1 gene was measured in three high oleic acid variants, namely S-317, Lesaff496, and CW99-OL, wild type The genotype SU was compared to that in the developing seed. As can be seen in FIG. 8, CtFAD2-1 expression is detected in all three stages of developing embryos in wild-type safflower genotype SU and has the highest level of expression observed in the middle maturation stage. In agreement with the existing results, a temporary transcription pattern of this major FAD2 gene was confirmed. However, CtFAD2-1 transcription was only slightly detectable in the three high oleic acid variants S-317, Lesaff496, and CW99-OL (FIG. 8), and high levels of RNA transcripts from this gene in mutant embryos The level of instability was shown.

  In contrast, the level of transcript from CtFAD2-2 is similar to wild type and high oleic acid genotype, CtFAD2-2 expression is not affected in HO embryos, and RNA preparations are assayed Preferably it was pure. Thus, CtFAD2-2 expression is also at a much lower level in wild-type seeds than for CtFAD2-1, but to Δ12-desaturation of fatty acids relative to storage lipids in developing safflower seeds. It was concluded that it could contribute and likewise be involved in Δ12-unsaturation of fatty acids relative to membrane lipids in roots, leaves and stems. Evidence that CtFAD2-2 expression increased in response to loss of CtFAD2-1 activity in developing safflower seeds of CtFAD2-1 mutants, or in contrast thereto, against high oleic acid mutations There wasn't.

The dramatically reduced CtFAD2-1 transcript in the HO strain is caused by non-sense mediated RNA degradation (NMD).
The dramatically reduced level of CtFAD2-1 transcripts in HO embryos is due to nonsense-mediated mRNA degradation of CtFAD2-1 mRNA because an immature stop codon was found in the middle of the coding sequence immediately after a single nucleotide deletion. (NMD) may have caused it. The NMD system is thought to be a mechanism involved in the degradation of abnormal mRNAs including immature stop codons (PTCs) that cause unexpected errors such as genomic mutations, transcriptional errors, and mis-splicing. It is a mechanism that exists widely in eukaryotes and has been extensively studied, especially in yeast and mammals. In higher plants, little has been studied, but the soybean Kunitz-type trypsin inhibitor gene (Kti3), the kidney bean-derived phytohemagglutinin gene (PHA) (Jofuku et al., 1989, Voelker et al., 1990). There are several reports, including the pea ferredoxin gene (FED1) (Dickey et al., 1994), and the rice waxy gene (Isshiki et al., 2001).

  In these experiments, it was shown that the ol mutation resulting in high oleic acid qualities in safflower seed oil correlated with low levels of CtFAD2-1 mRNA accumulation in developing seeds. Previous studies have shown that the ol allele is semi-recessive and is not consistent with the post-transcriptional gene silencing mechanism mediated by small RNAs. Gene silencing involves 21-24 nt siRNA generated from double-stranded RNA, results in transcription of antisense or hairpin RNA, and can genetically act as a locus for dominant or semidominant genes (Brodersen and Voicenet, 2006). To confirm that the mechanism of ol mutation is different from RNAi-related gene silencing, small RNA sequencing was performed as follows.

  Two small RNA libraries derived from high oleic acid genotype S-317 and wild type SU were generated using pooled RNA isolated from metaphase mature developing embryos. Bulk sequencing of small RNA libraries was performed using Solexa technology (Hafner et al., 2008). These two libraries were sequenced on a Solexa Sequencer from Illumina and samples were run side by side. Sequencing of the SU and S-317 small RNA libraries resulted in a total of 23,160,261 and 21,696,852 raw readings, respectively. Analysis of these readings resulted in the identification of 22,860,098 and 21,427,392 sequences, respectively, ranging in length from 18-30 nucleotides (nt). The presence and dispersion of small RNAs corresponding to CtFAD2-1 in the SU and S-317 libraries were determined. Only low and almost undetectable levels of small RNA corresponding to CtFAD2-1 were detected from both small RNA libraries and distributed almost evenly over the coding region of the CtFAD2-1 gene. There was no obvious difference between the wild type library and the high oleic acid library.

  From this data, it was concluded that small RNA-mediated post-transcriptional gene silencing prevented the accumulation of transcripts of mutant CtFAD2-1 but not the major mechanism.

N. To further investigate the transient expression study NMD phenomenon in Ventamiana leaves, we Experiments were conducted on the transient expression of CtFAD2-1 from both wild-type and high oleic acid genotypes in bentamiana leaves.

  Each of the CtFAD2-1 ORFs was inserted into the pORE04 binary vector modified in the sense direction under the control of the CaMV-35S promoter. Agrobacterium tumefaciens strain AGL1 harboring either 35S: CtFAD2-1 or its mutant form 35S: CtFAD2-1Δ was isolated from fully expanded N. cerevisiae with 35S: P19 described in Example 5. Invaded the back of the bentamiana leaf. After growing for an additional 5 days at 24 ° C., the infiltrated area was excised and total RNA was obtained from the samples using the Rneasy mini kit (Qiagen). To measure CtFAD2-1 RNA levels, a real-time qPCR assay was performed in triplicate using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) as described in Example 1, and the ABI 7900HT sequence detection system Operated above. PCR was initially performed at 48 ° C. for 30 minutes, then at 95 ° C. for 10 minutes, followed by 95 ° C. for 15 seconds and 60 ° C. for 60 seconds under 40 cycles. The primers for the exogenous CtFAD2-1 gene were sense: 5'-GTGTATGTTCGCCCTCGAGA-3 '(SEQ ID NO: 146) and antisense: 5'-GCAAGGTAGTAGAGACGAAG-3' (SEQ ID NO: 147). The reference gene, safflower CtKASII was used to normalize the expression level and its specific primers were sense: 5′-CTGAACTGCAATTATTTAGGG-3 ′ (SEQ ID NO: 144) and antisense: 5′-GGTATTGGTATTGGATGGGGCG-3 ′ (sequence No. 145). N. In Ventamiana leaves, a high level of CtFAD2-1 expression was observed from the 35S-CtFAD2-1 gene derived from the wild-type SU variant. In contrast, a much lower level of expression was observed for the 35S-CtFAD2-1Δ gene from the high oleic acid genotype.

  According to the method of Clow and Bent (1998), A. A saryana ecotype Col-0 plant has a binary vector with a seed-specific promoter Fp1 inside that activates the coding region of either CtFAD2-1 or CtFAD2-1Δ. It was transformed with Tumefaciens AGL1. Total RNA was isolated from long-horned fruit using the Rneasy mini kit (Qiagen) containing mid-stage embryos of the resulting transformed plant progeny. Gene expression studies were performed by real-time RT-qPCR assay using RNA preparations and performed in triplicate as described above. A high level of CtFAD2-1 expression was observed in Arabidopsis longhorn fruits expressing SU-derived Fp1-CtFAD2-1, but the expression of Fp1-CtFAD2-1Δ derived from a high oleic acid genotype is Decreased.

  Although transcripts of mutant CtFAD2-1 were dramatically reduced in total, small RNA specific for CtFAD2-1 was found in developing high oleic safflower seeds compared to small RNA from the wild type gene. It was shown that it was not produced at a significantly higher level. Therefore, we concluded that the decrease in CtFAD2-1 RNA in the high oleic acid genotype is due to NMD and is different from the gene silencing mechanism after small RNA-mediated transcription. Also, the NMD phenomenon is that the mutation coding region is N.P. It was observed when expressed exogenously in either benthamiana leaves or arabidopsis longhorns.

Example 8 FIG. Isolation of safflower cDNA, a candidate for encoding FATB
Isolation of Safflower FATB cDNA Sequence Safflower seed oil contains approximately 7% palmitic acid. This fatty acid is synthesized in the plastid of the developing seed cell and from there it is exported to the cytosol of the cell for its incorporation into triacylglycerol. The main enzyme for palmitic acid export is palmitoyl-ACP thioesterase, which hydrolyzes the thioester bond between the palmitoyl moiety and the acyl carrier protein (ACP), which covalently binds the acyl group, Synthesized with plastids. The enzyme palmitoyl-ACP thioesterase belongs to an enzyme that targets a series of soluble plastids, designated FATB. In seed oil plants, this enzyme exhibits specificity for short-chain saturated acyl-ACP as a substrate. First, the gene encoding the FATB enzyme was isolated from a plant species that accumulates medium chain length saturated fatty acids such as lauric acid (C12: 0) from California laurel (Umbellaria californica). Subsequent studies have shown that FATB homologous species are present in all plant tissues, primarily in seeds, and have substrate specificity ranging from C8: 0-ACP to C18: 0-ACP. In most warm oil seed crops including Arabidopsis and safflower, palmitic acid is the major saturated fatty acid in seed oil.

  To isolate safflower cDNA encoding a candidate for FATB, as described in Example 1, a growing safflower seed cDNA library was prepared using a FATB from gossipium hirstum. Screening was performed using a heterologous probe consisting of a cDNA fragment. One full-length cDNA, designated CtFATB-T12, was isolated from a safflower seed cDNA library. This cDNA contained a 1029 nucleotide long open reading frame encoding a 343 amino acid polypeptide. The 5 'and 3' UTRs were 236 nt and 336 nt lengths, respectively. The CtFATB-T12 polypeptide had a predicted transport peptide of approximately 60 amino acids and a 210 amino acid residue core containing two repeats of helix and multi-strand sheet folding, generally a so-called hot dog folding protein.

  From the expression sequence tag (EST) database (cgpdb.ucdavis.edu/cgpdb2) of the Composite Genome Project (CGP) for safflower, three different ESTs with homology to CtFATB-T12, namely EL379517, EL389828, And EL396749 were identified. Each was part length. The corresponding genes were designated CtFATB-A, CtFATB-B, and CtFATB-C, respectively. Full-length cDNA CtFATB-T12 isolated from safflower seed cDNA library was identified in the nucleotide sequence from CtFATB-C to EST in their overlapping region. CtFATB-A appeared to be more branched in its nucleotide sequence compared to the other two CtFATB sequences.

CtFATB gene expression profiles by real-time qPCR analysis As outlined in Example 1, gene expression profiles of three CtFATB genes were studied using real-time qPCR. Oligonucleotide primers were designed corresponding to each unique region of the three genes, including CtFATB-A, sense primer: 5'-AGAGATCATTGGAGATAGTAGAGTG-3 '(SEQ ID NO: 148); antisense primer: 5'- CCCATCAAGCACAATTTCTTCTTAG-3 ′ (SEQ ID NO: 149); CtFATB-B, sense primer: 5′-CTACACAATGCGACTCTGGTGCT-3 ′ (SEQ ID NO: 150); antisense primer: 5′-GCCATCCATGACACTATTTCTA-3 ′ (SEQ ID NO: 151); , Sense primer: 5'-CCTCACTCTGGGACCAAGAAAT-3 '(SEQ ID NO: 152); antisense primer: 5'-TTCTTG GACATGTGACGTAGAA-3 'is (SEQ ID NO: 153) were included. As described in Example 1, PCR reactions were performed in triplicate.

  As shown in FIG. 9, CtFATB-A showed low expression levels in all three stages of tested leaves, roots, and developing embryos. CtFATB-B was active in leaves and roots, but showed lower expression in developing embryos than in leaves and roots. This suggested that this gene, if any, could only play a minor role in fatty acid biosynthesis in developing seeds. In contrast, CtFATB-C showed high expression levels across all tissues tested, especially in the developing embryo. This indicated that CtFATB-C was the major gene encoding FATB for the production of palmitic acid in safflower seed oil. This was consistent with the recovery of only one FATB cDNA clone from the seed embryo library, namely CtFATB-T12, and was identical to the sequence for CtFATB-C. Based on these data, an approximately 300 bp DNA fragment from CtFATB-T12 (CtFATB-C) was used to prepare an hpRNA construct for downregulation of FATB in safflower seeds, as described in the Examples below. Selected as the gene sequence used in

Example 9: Isolation and expression of safflower cDNA encoding FAD6
Isolation of safflower FAD6 cDNA sequence The distinctly different fatty acid composition found in microsomes and chloroplast membrane lipids and seed storage oils is a complex metabolism that manipulates this composition by regulating fatty acid biosynthesis. As a result of the network, it flows through both so-called prokaryotic and eukaryotic pathways. It is clear that the microsomal FAD2 enzyme has an important role in converting oleate to linoleate in the ER after export of oleate to oleate and conversion to CoA ester in the cytoplasm. Chloroplast omega-6 desaturase (FAD6) contains all 16: 1- or 18: 1-, including phosphatidylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoginovosyldiacylglycerol, respectively. It is an enzyme that desaturates 16: 1 and 18: 1 fatty acids to 16: 2 and 18: 2 on chloroplast membrane lipids. The Arabidopsis fad6 mutation has been reported to be deleted on 16: 2 and 18: 2 desaturation of 16: 1 and 18: 1 on all chloroplast lipids, respectively (Browse et al. al., 1989). When the fad6 mutant was grown at a low temperature (5 ° C.), the leaves were bleached and the growth rate was markedly reduced compared to the wild type (Hugly and Somerville, 1992). The cDNA sequence encoding FAD6 was first described in Falcon et al. (1994) isolated from Arabidopsis. Since then, cDNAs encoding FAD6 and FAD6 genes have been isolated from several plant species including brush kanapus, portula caoleracea, soy, and lisinus communis.

  To isolate a cDNA clone encoding the chloroplast ω6 desaturase encoded by the safflower-derived FAD6 gene, the CPG database for homologous sequences was searched. Eight EST sequences, namely EL378905, EL380564, EL383438, EL385474, EL389341, EL392036, EL393518, EL411275, were identified and assembled into a single 808 nt contig sequence. This sequence had an intact 5 'end but was incomplete at the 3' end. Subsequently, full-length cDNA was obtained through 3'RACE PCR amplification using DNA extracted from a lambda cDNA library made from developing seeds of safflower (SU) as a template. PCR conditions were as described in Example 2. A single oligo primer designated ctFAD6-s2 was used in the amplification reaction in combination with the M13 forward primer because the sequence of this primer was present in the vector of the cDNA library. The sequence of the ctFAD6-s2 primer was 5'-CATTGAAGTCGGTATTGATATCTG-3 '(SEQ ID NO: 154). A 1545 bp cDNA with a 1305 bp open reading frame encoding a 435 amino acid candidate FAD6 polypeptide was obtained. This polypeptide shared 60-74% amino acid sequence identity with other cloned plant FAD6 polypeptides. A phylogenetic tree showing the phylogenetic relationship between the safflower FAD6 sequence and a representative FAD6 plastid Δ12 desaturase identified in higher plants was formed by the vector NTI (FIG. 10).

Expression profile of CtFAD6 by real-time qPCR analysis The expression profile of CtFAD6 was examined by real-time RT-qPCR performed using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and defaulted as described in Example 1 The parameters were used to operate on an ABI 7900HT sequence detection system. The primers used were ctFAD6-S2: 5′-CATTGAAGTCGGTATTGATATCTG-3 ′ (SEQ ID NO: 155) and ctFAD6-a2: 5′-GTTCCAACAATATCTTCCACCAGGT-3 ′ (SEQ ID NO: 156). The reaction was performed in triplicate in a total volume of 10 μL containing 20 ng total RNA template, 800 mM each primer, 0.25 μL reverse transcriptase, and 5 μL one-step RT-PCR master mix reagent. The conditions for RT and amplification were 48 ° C. for 30 minutes, then 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 60 seconds. Expression of the reference gene safflower CtkasII was used to normalize FAD6 expression levels. Calculations were performed as described in Example 1.

  This analysis indicated that CtFAD6 was expressed at relatively low levels in three successive stages of leaves, roots, and developing embryos. The low expression levels observed in developing seeds were consistent with the idea that FAD6 may have a relatively small role in desaturation of oleate in seeds.

Example 10 Design and preparation of genetic constructs for silencing fatty acid biosynthetic genes in safflower Hairpin RNA (hpRNA) is a type of RNA molecule that is widely used to reduce gene expression in plants. Hairpin RNA is generally transcribed in a plant cell from a DNA construct containing an inverted repeat sequence derived from the gene to be silenced. The hpRNA transcript thereby has complementary sense and antisense sequences that hybridize to form a double stranded RNA (dsRNA) region joined by a loop sequence. Such dsRNA structures are processed in plant cells by an endogenous silencing mechanism to form small RNA molecules of about 21-24 nucleotides corresponding to genes whose activity is reduced in sequence. These small RNAs can form complexes with endogenous proteins that specifically silence the gene of interest. Such silencing is at the transcriptional level mediated by DNA methylation of the target gene portion, at the post-transcriptional level due to degradation of the target mRNA, or binds to that mRNA, inhibits its translation, and thereby by the gene. This can occur by reducing the encoded protein synthesis. If the hpRNA contains a sequence that is common among members of the gene family, the hpRNA can be silenced to each of these genes having the sequence.

  Safflowers have a large family of FAD2 genes (Examples 2-6) with at least 11 members identified as described herein. The safflower also has multiple genes (Example 8) that encode a FATB polypeptide with at least three members identified, and at least one FAD6 gene (Example 9). In fact, the above experiments may not have identified all members of the gene family in safflower. To determine which members of the FAD2 and FATB gene families were involved in the biosynthesis of linoleic or saturated fatty acids found in safflower seed oil, particularly palmitic acid, and whether FAD6 was also involved, Genetic constructs expressed hpRNA molecules in safflower and silenced various combinations of FAD2, FATB, and FAD6 genes. Each of these hpRNA constructs was designed to be specifically expressed in developing safflower seeds during oil synthesis. This was done by using either a foreign promoter or a promoter isolated from safflower to express the construct, which was introduced into safflower by plant transformation.

Construction of pCW600 A plant binary expression vector was designed for the expression of the transgene in seeds and used the promoter (SEQ ID NO: 52) of the Arabidopsis Olesoin1 gene (TAIR website gene annotation At4g25140). The isolated promoter is 1192 bp long and started at nucleotide 12899298 with accession number NC003075.7, but within the 1198 bp sequence to avoid normal restriction digestion sequences and to assist in subsequent cloning steps. 6 bp was deleted. The AtOleosin promoter was previously used for strong seed-specific expression of transgenes in safflower and Brassica species (Nykiforuk et al., 1995, Vanrouijen and Moloney, 1995). This promoter was likely a bidirectional promoter that induces strong seed-specific expression of the coding region bound to both ends of the promoter fragment. The Arabidopsis oleosin promoter shares characteristics with the brush kanapus oleosin promoter, which is characterized by having bifunctionality (Sanadanom et al., 1996). This promoter was cloned into an EcoRI fragment containing a promoter that was chemically synthesized and blunted by pGEMT-Easy and Klenow fragment enzyme packing reactions and blunted by Klenow of pCW265 (Belide et al., 2011). Ligation to the HindIII site generated pCW600 (AtOleosinP :: empty). This vector has a selectable marker gene encoding hygromycin phosphotransferase (HPT), thereby allowing selection for resistance to hygromycin in tissue culture during the transformation process. did. This vector also contained a 35S :: GFP gene that allowed selection of fluorescently transformed cells or tissues under UV irradiation. By inserting the AtOleosin promoter, this vector is designed for the expression of a coding region of interest that can be inserted downstream of a promoter located at multiple cloning sites and upstream of a nos polyadenylation signal (nos 3 ′). It was done. This vector served as the backbone vector for the constructs pCW602 and pCW603 described below.

Construction of pXZP410 Flax linin promoter (US Pat. No. 7,642,346) (SEQ ID NO: 53) was inserted as NotI-XhoI fragment into binary vector pT7-HELLSGATE12 (Wesley et al., 2001) for Gateway silencing The binary vector pXZP410 was generated. Vector pXZP410 has a selectable marker gene that confers resistance to kanamycin in tissue culture, allowing seed-specific expression of the hairpin RNA construct under the control of the linin promoter. This vector had two introns, one in the sense orientation and one in the antisense orientation, relative to the promoter, and had AttL1 and AttL2 recombination sites adjacent to the intron.

  To create a silencing construct from pXZP410, two copies of the sequence from the target gene in the Gateway invasion vector to be silenced are inserted into the vector and one of two introns in the opposite orientation relative to each other. Was inserted 5 'and the other was inserted 3' to form inverted repeats. The recombination site, as described above, facilitated the insertion of two copies by using the Gateway recombinase cloning system (Invitrogen, Carlsbad, USA) (Wesley et al., 2001). This vector pXZP410 was used as the backbone vector to generate pCW631 and pCW632 as described below.

Construction of pCW571 A 300 bp sequence (SEQ ID NO: 50) identical to the region of the safflower gene CtFATB-3 corresponding to nucleotides 485-784 of the CtFATB-3 cDNA was chemically synthesized and according to the manufacturer's instructions, A Gateway invading clone was generated which was inserted into pENTR / D topo (Invitrogen) and designated pCW569. A 756 bp fragment of the CtFAD2-2 cDNA corresponding to nucleotides 427-1182 (SEQ ID NO: 49) was synthesized and amplified by RT-PCR from RNA isolated from developing safflower seeds. The primers were D28-PstI-5 (5′-CCTGCAGGTACCAATGGCTCGACGACACTG-3 ′) (SEQ ID NO: 157) and D28-AscI-3 (5′-CGGCCGCGCTTCACCCTCCCTCATTTTTCC-3 ′) (SEQ ID NO: 158), respectively. The primer contained 5 'PstI and 3' AscI restriction enzyme sites so that the amplified fragment could be inserted into the corresponding site of pCW569, generating pCW570. This vector contained the fusion region from the CtFATB and CtFAD2-2 genes as a 1080 bp fragment flanked by recombination sites AttL1 and AttL2. Two copies of this FATB-FAD2-2 fragment were then inserted into pXZP410 and the second copy was used as the first copy using LR clonase (Invitrogen, Carlsbad, USA) according to the supplier's instructions. Inserted into. The resulting plasmid, pCW571, has a flax linin promoter to transcribe the inverted repeat region in a seed-specific manner to generate hpRNA to reduce the expression of CtFATB and CtFAD2-2 genes in the seed. did.

Construction of pCW603 A DNA fragment from pCW571 containing an inverted repeat CtFATB-CtFAD2-2 fragment with two intervening introns was cut with SpeI, blunted using the Klenow I fragment of DNA polymerase, and the EcoRV of pCW600. Ligated to the site, pCW603 was generated. This construct, pCW603, was able to express hpRNA under the control of the AtOleosin promoter and reduce the expression of CtFATB and CtFAD2-2 in safflower seeds.

Construction of pCW581 For pCW571, a 290 bp fragment of DNA (SEQ ID NO: 51) made from a 290 bp fragment of CtFAD6 and a 300 bp fragment of CtFATB corresponding to nucleotides 451-750 of the cDNA of CtFAD6 was chemically synthesized. Inserted into pENTR / D topo to generate pCW579. As described above for pCW570, a 780 bp fragment of CtFAD2-2 was cloned into the AscI site of pCW579 to generate pCW580. This construct was an invading clone vector containing sequences joined in the order of the CtFATB, CtFAD6, and CtFAD2-2 genes as a 1370 bp DNA fragment with adjacent recombination sites AttL1 and AttL2. Two copies of this FATB-FAD6-FAD2-2 fragment were then inserted into pXZP410 as inverted repeats using LR clonase to generate pCW581. This construct, pCW581, is a binary vector with a flax linin promoter operably linked to inverted repeats, and during transcription of developing safflower seeds, cells reduce the expression of CtFATB, CtFAD6, and CtFAD2-2 genes In order to express hpRNA.

Construction of pCW602 A DNA fragment containing the inverted repeat coupled CtFATB-CtFAD6-CtFAD2-2 region with two intervening introns is enzymatically cleaved from pCW571 with Not1 and blunted using the Klenow I fragment And then ligated to the EcoRV site of pCW600 to generate pCW602. In contrast to pCW581, which had the same design and gene fragment except that the linin promoter was used, pCW602 had a CtFATB-CtFAD6-CtFAD2-2 sequence under the control of the AtOleosin promoter.

Construction of pCW631 and pCW632 The linin promoter was useful for expressing hpRNA in seeds, but both pCW571 and pCW581 had selectable markers conferring kanamycin resistance. In preliminary safflower transformation experiments, we observed that explants were not fully affected by kanamycin. Therefore, the kanamycin resistance cassette of pCW571 and pCW581 was replaced with a hygromycin resistance cassette as a selectable marker gene. The hygromycin resistance gene consisting of the enCUP promoter: hygromycin: nos3 ′ polyadenylation region was cleaved from pCW265 with SpeI-AvrII restriction digests and used in pCW571 and pCW581 to replace the kanamycin resistance cassette, respectively, pCW631 and 632 was produced.

In summary, the construct used for this first set of safflower transformation included the following major elements:

Gene fragments with vector promoter inverted repeats
pCW631 Linine CtFAD2-2 and CtFATB
pCW632 Linine CtFAD2-2, CtFAD6, and CtFATB
pCW602 AtOleosin CtFAD2-2, CtFAD6, and CtFATB
pCW603 AtOleosin CtFAD2-2 and CtFATB

  These constructs were introduced into Agrobacterium strain AGL1 and used to transform safflower as described in Example 1 with the following results.

Example 11 Transformation of safflower with gene silencing constructs The genetic construct transformed the safflower varieties S317 excised cotyledons and hypocotyls using an Agrobacterium method to rescue buds regenerated using grafts. Used to convert (Belide et al., 2011). Non-transformed rootstock (hereinafter, T ₀ termed plants) Separate shoots transformed with more than 30 that are growing on may be played to the vector PCW603, described in Example 1 So as to mature. Integration of T-DNA in T ₀ safflower shoots is described in Belide et al. (2011) as confirmed by PCR using primers specific for the T-DNA vector. Most plants that were found to lack the T-DNA used for specific transformations and were supposed to be “escapes” from hygromycin selection were discarded during regeneration in tissue culture. However, some were maintained as “null” plants or negative controls for comparison with transformed plants. These control plants were treated under the same conditions, grafting, and greenhouse conditions as the material transformed in tissue culture.

Example 12 Analysis fatty acid analysis of the fatty acid composition of seed oil of the transgenic safflower is as follows, were made in individual T ₁ seeds obtained from safflower plants that have been transformed. In S317 genetic background, 30 of each _{T 0} plants transformed with pCW603 is, are grown in the greenhouse, and self-fertilized to produce seeds. As many as 10 mature seeds from the head of a single seed from each T ₀ plant were analyzed for lipid composition using GC analysis as described in Example 1. The results of fatty acid composition analysis from safflower S317 seeds transformed with pCW603 are summarized in Table 13. T ₀ safflower plants each transformation is a heterozygous for T-DNA, therefore, because it was expected to produce T ₁ seeds segregating population, 5 to 10 seeds from each plant It was predicted that the analysis could include several null seeds. The seeds of such null segregants were good negative controls in this experiment because they grew and developed in the same seed head as transformed seeds from the same plant. As can be seen from the data in Table 13, the level of oleic acid above 87% (as weight% of total fatty acid content) was increased to 6 independent strains (strains 9, 12) out of 30 produced strains. , 14, 20, 34, and 36). Many transformed seeds have an oleic acid content ranging from 87-91.7%, a linoleic acid level of 2.15 to 5.9% and a palmitic acid level of 2.32 to 3.45% Had. Other fatty acid levels in seeds were not significantly different from non-transformed controls. The maximum oleic acid content observed in T ₁ safflower seeds transformed with pCW603 was 91.7%, compared to about 77% in non-transformed S317 control seeds and null isolate seeds. Met. Notably, seed lipids also decreased significantly at the 16: 0 level, decreasing from 4.5% to 2.3%. When purified on TLC plates, the fatty acid profile of the TAG fraction of seed oil was not significantly different from that of total lipids extracted from seeds.

  Two metrics were calculated based on the total fatty acid composition of safflower seeds that accounted for the most important fatty acids in safflower seed oil. These were the oleic acid unsaturation (ODP) and the ratio of palmitic acid + linoleic acid to oleic acid (PLO). These are calculated for each seed and this data is shown in Table 13. Wild type seeds (S317, non-transformed) and null segregants had an ODP ratio of about 0.1500 and a PLO value of about 0.2830. Seeds transformed with T-DNA from pCW603 showed a significant decrease in ODP and PLO values. Thirteen seeds resulting from six independent events had PLO values less than 0.1 and ODPs less than 0.06. One transformed strain had 0.0229 ODP and 0.0514 PLO.

  One elite strain of mature individual single seed, S317 transformed with strain 9 of pCW603, and untransformed parent S317 were subjected to analysis of lipid total metabolites in LC-MS. These analyzes clearly showed that oil from seeds transformed with the AtOleosinp: CtFATB-CtFAD2-2 RNAi hairpin construct dramatically changed TAG and DAG composition (FIGS. 11 and 12). There is a clear increase in the level of TAG (54: 3) and a decrease in TAG (54: 5), which are mainly 18: 1/18: 1/18: 1-TAG (triolein) and 18 respectively. : 1/18: 2/18: 2-TAG. Of the seeds analyzed by LC-MS, the triolein (= 54: 3) content in TAG was 64 at the highest oleic acid level (over 90%, Table 14) relative to seeds from RNAi silencing lines. Equivalent to .6% (mol%), whereas non-transformed (S-317) parents had triolein levels ranging from 47% to 53%. The TAG containing the second most abundant oleate was 18: 0/18: 1/18: 1 followed by 18: 1/18: 1/18: 2. Also, the most obvious differences between these safflower oils could be seen in the DAG lipid class. DAG (36: 1) levels were doubled with seed oil from transformed seeds compared to the parent seed, but DAG (36: 4) was reduced by up to 10%. DAG (36.1) is mainly 18: 0/18: 1-DAG, and DAG (36: 4) is 18: 2/18: 2-DAG (dilinoleate). Since the level of total TAG was about 100 times higher than total DAG, DAG in seed lipids was a slight component (Table 15).

_{T 0} Safflower transformed cells containing T-DNA of the growth and morphology from pCW603 transgenic plants, generates a _{T 1} seeds separated from the T-DNA, a set of homozygotes, heterozygotes And null isolates were obtained. The ratio of these sub-populations, as would be expected by Mendelian genetics, different according to the number and connection of the T-DNA insertion events in T ₀ plants. Therefore, T ₁ seeds from each T ₀ plant were analyzed independently. Analysis of lipid profiles from individual seeds clearly showed that a single seed head contained both null and transgenic events.

Seeds from some transgenic lines grow under controlled conditions in the greenhouse (temperature, soil, optimal watering and fertilization but under natural light) to observe plant morphology and growth rate It was done. No phenotypic differences were observed between the transformed T ₁ plants and their sibling siblings. The transformed seeds germinated at the same rate as the non-transformed seeds to obtain seedlings having the same early seedling growth rate (growth potential). All planted seeds were indigenous and grew into fully fertilized plants. DNA is prepared from the tips of the leaves from the T ₁ generation of individual plants, PCR analysis was performed to determine the null and ratios of the transgenic plants. As expected, a null segregant was identified.

  This phenotypic analysis showed that safflower plants transformed with pCW603 T-DNA and expressing the transgene did not suffer any adverse effects compared to null segregants.

  T2 generation safflower seeds were tested for oil composition. Some plant-derived seeds (Table 13) with high levels of oleic acid grew into mature plants producing second generation seeds (T2 seeds). These seeds were harvested when matured and analyzed for the fatty acid composition of their oil. This data is shown in Table 16. T1 seeds showed a maximum of about 92% oleic acid, while T2 seeds reached 94.6% oleic acid. The increase observed in the T2 generation relative to the T1 generation can be due to the homozygosity of the transgene or simply because a large number of strains have been analyzed.

  Southern blot hybridization analysis is used to determine the number of T-DNA insertions in each transformed strain, and strains with a single T-DNA insertion are selected. The oil content of T2 seeds is not significantly different from those grown under the same conditions in controlled non-transformed seeds of the same genetic background.

Analysis of safflower seeds transformed with T-DNA from pCW631 T1 seeds of safflower varieties S-317 transformed with pCW631 were similarly analyzed for their fatty acid composition. Table 17 shows the data for these seeds and the ODP and PLO metrics. The oleic acid content in the lipids of these seeds was a maximum of 94.19%. Seed TS631-01 T1 (21) with the highest level of oleic acid had a palmitic acid content of 2.43%, an ODP of 0.0203, and a PLO of 0.0423. These analyzes indicated that the pCW603 construct, the pCW631 hairpin RNA construct, generally produced higher oleic acid levels when transformed into the S-317 genetic background safflower. This observation indicates that the linin promoter used in pCW631 expressed a hairpin RNA with a stronger or better timing of expression, or a combination of both, compared to the AtOleosin promoter used in pCW603. It was.

  T2 seeds of safflower variant S-317 transformed with T-DNA from pCW631 were analyzed by GC. Up to 94.95% oleic acid levels were observed with 0.01 ODP and 0.035 PLO.

  Safflower seeds and plants transformed with T-DNA from the constructs pCW632 and pCW602 were analyzed in the same way as for seeds and plants transformed with pCW603 and pCW631. GC analysis of T1 seeds of safflower variant S-317 transformed with pCW632 showed an oleic acid level of up to 94.88% with 0.0102 ODP and 0.0362 PLO. Those T2 seeds showed up to 93.14% oleic acid with 0.0164 ODP and 0.0452 PLO.

Extraction of larger amounts of safflower seed oil T4 seeds from a homozygous transgenic strain designated TS603-22.6 are harvested and the whole seed oil is used in a Soxhlet apparatus as described in Example 1 Extracted. An aliquot of the extracted oil was analyzed by GC (Table 18). A total of 643 grams of oil was recovered. Extracts 2, 3, 5, and 6 were pooled, while extracts 4, 7, and 8 were pooled in separate lots. These mixtures were further analyzed for fatty acid composition by GC. This data is shown in Table 18.

Example 13 Design and Preparation of Additional Gene Silencing Constructs Based on the results described in Examples 10-12, other gene silencing constructs were prepared as follows to increase the oleic acid content of safflower seed oil and to increase ODP Or the PLO ratio was decreased. These gene silencing constructs combine non-safflower sources and different promoters from safflower sources to achieve maximal reduction in safflower FAD2-2, FATB-3, and FAD6 gene expression, and FAD2-2. In addition to further silencing more than one FAD2 gene. These constructs were used to transform safflower varieties with inactive variants of the endogenous CtFAD2-1 gene, eg, S-317, Ciano-OL, and Lesaff496.

Construction of pCW700 This plant binary expression vector differs in safflower seeds, but rather in a safflower seed, rather than a single promoter that produces hairpin RNA to reduce the expression of endogenous CtFATB and CtFAD2-2. Have two foreign (non-safflower) promoters. The two promoters are the AtOleosin promoter and the flax linin promoter, and the two hpRNA expression cassettes are in the same T-DNA molecule. This vector was constructed by restriction digestion of the hpRNA gene expression cassette from pCW631 with the linin promoter and the hpRNA coding region for silencing CtFAD2-2 and CtFATB, which was inserted into the T-DNA from pCW603, Thus, constructs are generated that encode hairpin RNAs for these two safflower genes. This construct is used to transform safflower varieties such as Lesaff496, Ciano-OL, and S-317.

Construction of pCW701-pCW710 The safflower-derived promoter expected to have optimal activity in safflower seeds uses DNA sequencing techniques that provide accurate sequence information about the upstream and downstream DNA regions of the expressed gene. Isolated. Previous results described in Examples 2-6 indicate that the CtFAD2-1 gene is highly expressed during seed development in safflower. Thus, the promoter region of this gene is an excellent candidate for activating efficient transgene expression in safflower seeds. As shown in Example 6, CtFAD2-2 was active in a genetic background where CtFAD2-1 was inactivated by mutation. Therefore, the promoter of CtFAD2-2 is used in safflower to activate the expression of hairpin RNA that targets CtFAD2-2 activity among other genes. Other promoter elements useful for transgene expression in safflower seeds include endogenous (ie, safflower) upstream portions of the genes for oleosin (CtOleosin) and seed storage proteins, eg, 2S and 11S proteins (Ct2S and Ct11S). ) Promoter elements are included. The CtFAD2-1, CtOleosin, Ct2S, and Ct11S promoter elements are isolated using standard PCR-based techniques based on safflower genomic sequences and incorporated into plant binary expression vectors. These promoter elements are used to construct hpRNA silencing molecules in the construct pCW701-pCW710 or in combination with other non-safflower promoters that express the same or different hpRNA genes, such as pCW602, pCW603, pCW631, or pCW632. To express. Combinations of hpRNA genes with different promoters are also generated by crossing transformed plants with individual genes, where hpRNA genes are generally not linked.

  To isolate the CtFAD2-1 promoter, a genomic DNA fragment approximately 3000 bp upstream of the CtFAD2-1 translation initiation ATG codon was isolated using a PCR-based approach to replace the AtOleosin promoter from pCW603 and pCW602 To generate constructs pCW701 and pCW702, respectively.

  To isolate the CtFAD2-2 promoter, a genomic DNA fragment approximately 3000 bp upstream of the CtFAD2-2 translation initiation ATG codon was isolated using a PCR-based approach and used to generate pCW603 and pCW602. Substituting the derived AtOleosin promoter to produce the constructs pCW703 and pCW704, respectively.

  To isolate the CtOleosin-1 promoter, a genomic DNA fragment approximately 1500 base pairs upstream of the CtOleosin translation initiation ATG codon was isolated using a PCR-based approach and replaced with the AtOleosin promoter from pCW603 and pCW602. To generate constructs pCW705 and pCW706, respectively.

  To isolate the Ct2S promoter, a genomic DNA fragment approximately 1500 base pairs upstream of the Ct2S translation initiation ATG codon was isolated and used to replace the AtOleosin promoter from pCW603 and pCW602, thereby Vectors pCW707 and pCW708 are generated, respectively.

  To isolate the Ct11S promoter, a genomic DNA fragment approximately 1500 base pairs upstream of the Ct11S initiation ATG was isolated from safflower genomic DNA using a PCR-based approach, and the AtOleosin promoter from pCW603 and pCW602. To generate the vectors pCW709 and pCW710, respectively.

  Each of these vectors is transformed into safflower varieties as described in Example 1.

Example 14 Field performance of safflower varieties A series of untransformed safflower varieties and accessions were grown in the summer of 2011-2012 on a field farm located in Narrabri, New South Wales. Seeds were planted on heavy clays commonly found in Narrabri regions within a 5m x 3m field plot. Plants were exposed to natural light and rainfall, except that they were irrigated once 4 weeks after growth. Mature seeds were harvested and approximately 50 seed samples were analyzed for lipid content and fatty acid composition in seed oil. The oleic acid content in the seed oil of various varieties and accessions is shown in Table 19 and FIG.

Data from field experiments surprisingly showed that there was a range of safflower seed oleic acid content within the observed range. Of note are various accessions described as “high oleic acid” that have been previously reported to provide seed oils with at least 70% oleic acid, such as Ciano-OL 42-46% oleic acid was produced. The level of linoleic acid was much higher than that predicted based on previous reports. In contrast, other accessions that have been reported to obtain high oleic acid content, such as, for example, Accession PI-5601698 and PI-560169, are actually high oleic acid levels in seed oil under field conditions. (60% -76%). Reasons for oleic acid levels that are significantly lower than expected in some accessions are due to the presence of non-ol alleles, eg, CtFAD2-1 alleles such as temperature-sensitive ol1 alleles, and 2011-12 It was thought to be related to growth conditions that were not very ideal during the year. Further fatty acid analysis in seeds obtained from field-grown safflowers was performed to confirm the varieties observed in the oleic acid content of several accessions.

  This experiment also shows that the level of oleic acid in seed oil obtained from plants grown in the field is generally higher than that from plants grown in the greenhouse, even for good field accessions. It was about 5-10% lower. This was thought to be because the conditions for growing outdoors were not as ideal as those in the greenhouse.

In further experiments, plants of safflower cultivar S317 were grown either in the field or in the greenhouse to compare the fatty acid composition of seed oil. The outdoor conditions were more favorable for the growing season compared to the 2011-12 period, but 20 from seeds grown in the field compared to 1.202 g from plants grown in the greenhouse. The seed weight was 0.977 g. 18-20 seeds of each group were analyzed for fatty acid composition by GC analysis. The oleic acid levels of seeds grown in the field ranged from 74.83 to 80.65 and had an average of 78.52 +/− 1.53 (+ / 1 standard deviation), whereas in the greenhouse The range of seeds grown was 75.15-78.44, with an average of 76.33 +/− 1.00. Other fatty acids were present at the levels in Table 20.

Example 15. Transgene crossing to other safflower varieties Safflower varieties are crossed manually using, for example, established methods as described in Mundel and Bergman (2009). The best transformed strains containing the above-mentioned constructs are selected, in particular transformed strains containing only a single T-DNA insert, and are not transformed with different constructs of other variants of safflower, Or crossed with already transformed plants with optimal agricultural productivity. For example, for 4 or 5 backcrosses, using a round of backcrosses with the recurrent parent, homozygous for the desired construct (s) in the genetic background of optimal agricultural productivity A self-propagating plant is produced. Marker-based selection can be used for breeding processes such as the use of complete markers for the ol allele, as described in Example 7.

Example 16 Modified microRNAs (miRNAs) of seed oil composition by gene silencing mediated by artificial microRNAs are a type of 20-24 nucleotides that are endogenous to both plants and animals that regulate the activity of endogenous genes (Nt) regulatory small RNA (sRNA). Transgenic expression of modified miRNA precursor RNA (artificial miRNA precursor) represents a recently developed RNA-based sequence-specific strategy for silencing endogenous genes. Substitution of several nucleotides in the miRNA precursor sequence to create an artificial miRNA precursor will result in repetitive generation of miRNA as long as the position of the match and mismatch remains unaffected within the stem loop of the precursor Has been shown to have no effect.

  The CSIRO software package MatchPoint (www.pi.csiro.au/RNAi) (Horn and Waterhouse, 2010) is unique when the Arabidopsis genome is unique and is therefore expressed when it is expressed as an artificial miRNA (off-gene targeting) ) Was used to identify specific 21-mer sequences in the Arabidopsis FAD2, FATB, and FAE1 genes that are unlikely to cause silencing. A unique 21-mer sequence was selected for each of the three genes, in which case the 21-mer was completely complementary (antisense) to the corresponding gene transcript region. It was. An artificial miRNA precursor molecule is Designed for each based on the precursor sequence of Sariana ara-miR159b. In each case, the precursor sequence of miR159b was modified in its trunk to match the antisense 21-mer sequence. Each of the three constructs encoding one of the precursor RNAs was made under the control of a seed-specific FP1 promoter, cloned into a binary expression vector and expressed as pJP1106, pJP1109, and pJP1110. Generated.

These constructs were separately isolated by electroporation A. The tumefaciens strain AGL1 was transformed, and the transformed strain was transformed into the genetic structure A.1 by the floral dip method. Used for introduction into Sariana (ecological type Columbia) (Example 1). Seeds from treated plants (T ₁ seed) in order to select the seedlings transformed, plan cultured on MS medium supplemented with PPT of 3.5 mg / L, transferred them to the soil, an established T ₁ transgenic plants were allowed to settle. Most of these T ₁ plants that are heterozygous was predicted for the introduced genetic constructs. T ₂ seeds from transgenic plants are collected at maturity and analyzed for their fatty acid composition. These T ₂ plants contained strains were homozygous, and what was heterozygous for the genetic construct. Homozygous T ₂ plants were self-fertilized to produce T ₃ seeds, and T ₃ progeny plants were obtained from these seeds, which were also used to obtain T ₄ progeny plants. This therefore enabled an analysis of the stability of gene silencing over three generations of progeny plants.

The fatty acid profiles of seed oils obtained from T ₂ , T ₃ , and T ₄ seed lots were analyzed by GC as described in Example 1. The change in Δ12-desaturase activity caused by the action of the FAD2-based transgene was seen as an increase in the amount of oleic acid in the seed oil profile. A related method for assessing the cumulative effect of Δ12-desaturase activity during seed fatty acid synthesis is through calculating the oleic acid unsaturation (ODP) parameter for each seed oil: ODP = 18: % Of 2 + 18: 3% / 18: 1% + 18: 2% + 18: 3%. Wild-type Arabidopsis seed oil generally has an ODP value of about 0.70 to 0.79, which is 70% to 79% of 18: 1 formed during fatty acid synthesis in the seed, after which First of all, it meant that 18: 2 was produced by the action of Δ12-desaturase and then converted to polyunsaturated C18 fatty acids by further desaturation to 18: 3. Thus, the ODP parameter is useful for determining the extent of FAD2 gene silencing at the level of endogenous Δ12-desaturase activity.

The level of C18: 1 ^Δ9 (oleic acid) in T ₂ seed transformed with the pJP1106 construct (FAD2 target) was 30% compared to the average level of wild type C18: 1 of 14.0% ± 0.2. In the range of 32.9% to 62.7% in the transgenic events. A highly silenced strain with a single transgene insertion (plant ID-30), determined by the segregation ratio (3: 1) of the plant selectable marker (PPT), is the next generation (T ₃ ). A similarly high level of 18: 1 ^Δ9 was observed in T ₃ seeds with an average of 57.3 ± 5.0%, ranging from 46.0% to 63.8%. In subsequent generations, _{T 4} seeds also in the range of 61% ～65.8%, with an average of 63.3 ± 1.06%, similarly high levels of 18: shows a 1 ^{[Delta] 9.} T ₂ total PUFA content of transgenic seed oil (18: 2 + 18: 3), which ranged from 6.1% to 38%, the total PUFA content of seed oil from homozygous lines ID-30 is further It decreased, and it was 4.3 to 5.7% of range. The control Arabidopsis ecological Columbia seed oil has an ODP value in the range of 0.75 to 0.79, which means that more than 75% of the oleic acid produced in the growing seed is then 18: 2 Or it was converted to 18: 3. In contrast, seed oil from the Arabidopsis fad2-1 mutant has an ODP value of 0.17, indicating a 75% reduction in Δ12-desaturation by the fad2-1 mutant. It was. ODP values are in the _{T 2} transgenic seed oil 0.08 to 0.48, 0.07 to 0.32 at _{T 3} seed oil, in the range of 0.06 to 0.08 at _{T 4} seed oil, it In contrast, the control Arabidopsis seed oil had a value of 0.75. The dramatic decrease in ODP values in the transgenic lines clearly demonstrated efficient silencing of the endogenous FAD2 gene using an artificial microRNA approach. This experiment also showed the stability of gene silencing over three generations. Similar degrees of gene silencing were seen with the other two constructs that down-regulated their corresponding genes.

The degree of FAD2 gene silencing and the amount of 18: 1 ^Δ9 (63.3 ± 1.06%) observed in this study using artificial microRNAs are well characterized FAD2-2 mutants Strains that silenced FAD2 (59.4%) and hairpin RNA approaches (56.9 ± 3.6%) and hairpin-antisense approaches (61.7 ± 2.0%). used. The mean 18: 2 + 18: 3% content in seed oil silenced with FAD2 using amiRNA was 4.8 ± 0.37%, which was previously reported for the FAD2-2 mutant Strains that were lower than those (7.5 ± 1.1%) and silenced FAD2 used the hairpin-antisense approach (7.2 ± 1.4%). Thus, these data showed the benefits of artificial microRNAs at the degree of silencing, as well as the stability of silencing across the generation of offspring.

Example 17. Assay of Sterol Content and Composition in Oil Plant sterols from 12 vegetable oil samples purchased from commercial sources in Australia, as described in Example 1, as GC derivatives as O-trimethylsilyl ether (OTMSi-ether) derivatives And characterized by GC-MS analysis. Sterols were identified by retention data, interpretation of mass spectra, and comparison with literature and experimental standard mass spectral data. Sterols were quantified by use of an internal standard of 5β (H) -cholan-24-ol. The basic plant sterol structure and chemical structure of some of the identified sterols are shown in FIG. 14 and Table 21.

  The analyzed vegetable oils are sesame (Sesamum indicum), olive (Orea Europaea), sunflower (Helianthus annuus), castor (Rishinus communis), rapeseed (Brush canapus), safflower (Cartamstinct) Derived from Rius), peanuts (Arakis hippogea), flax (Linham beef tacitimam), and soybeans (Glycine max). The major plant sterols were reduced in relative abundance across all of the oil samples: β-sitosterol (range 28-55% of total sterol content), Δ5-avenasterol (isofucosterol) (3-24%) , Campesterol (2-33%), Δ5-stigmasterol (0.7-18%), Δ7-stigmasterol (1-18%), and Δ7-avenasterol (0.1-5%) there were. Several other secondary sterols have been identified, these were cholesterol, brassicasterol, carinasterol, campesterol, and everychol. Four C29: 2 and two C30: 2 sterols were also detected, but further studies are needed to complete the identification of these subcomponents. In addition, some other unidentified sterols were present in some oils, but due to their very low abundance, the mass spectra were not strong enough to allow their structure to be identified.

  Sterol content expressed as decreasing amounts of mg / g (oil) is rapeseed oil (6.8 mg / g), sesame oil (5.8 mg / g), flax oil (4.8-5.2 mg / g) , Sunflower oil (3.7-4.1 mg / g), peanut oil (3.2 mg / g), safflower oil (3.0 mg / g), soybean oil (3.0 mg / g), olive oil (2.4 mg / G), castor oil (1.9 mg / g). The sterol composition and total sterol content (%) are shown in Table 22.

  Of all the seed oil samples, the major plant sterol was generally β-sitosterol (range 30-57% of the total sterol content). There were various oils in proportions of other major sterols: campesterol (2-17%), Δ5-stigmasterol (0.7-18%), Δ5-avenasterol (4-23%), Δ7 -Stigmasterol (1-18%). Oils from different species had different sterol profiles than those with completely different profiles. Rapeseed oil had the highest proportion of campesterol (33.6%), while other species samples generally had lower levels, eg, up to 17% in peanut oil. Safflower oil had a relatively high proportion of Δ7-stigmasterol (18%), which was usually low in other types of oil and up to 9% in sunflower oil. Since they were unique in each species, the sterol profile was therefore to help identify specific plants or vegetable oils and to confirm the addition of impurities by their pure species or other oils Can be used.

Two samples of sunflower and safflower were each compared, in which case one was produced by cold compression of the seed and was unpurified, while the other was not cold compressed and purified. Although some differences were observed, the two sources of oil had similar sterol composition and total sterol content, suggesting that processing and refining had little effect on these two parameters. The sterol content in the sample varied 3 fold and ranged from 1.9 mg / g to 6.8 mg / g. Rapeseed oil had the highest sterol content and castor oil had the lowest sterol content.

  Separate analyzes were performed on safflower oil from greenhouse-produced control seeds (S series), genetically modified high oleic acid seeds (T series), and two commercial safflower oils. Several features were observed (Table 23). First, there is a high degree of sterol pattern similarity between control seeds and modified seeds, and second, commercially available safflower oil is grouped separately and hence significantly different plant sterols. It has been shown to have a profile. Further testing of plant sterol profiles also showed plant sterol profile similarity from control and modified safflower seed samples.

  It will be apparent to those skilled in the art that various changes and / or modifications to the invention as illustrated in the specific embodiments can be made without departing from the spirit or scope of the invention as broadly described. Therefore, it should be thought that embodiment of this invention is an illustration and restrictive at no points.

  This application claims priority from US Patent No. US61 / 638,447 filed on April 25, 2012, and Australian Patent No. AU2012903996 filed on September 11, 2012, which are Both are incorporated herein by reference.

  All publications discussed and / or mentioned herein are incorporated herein in their entirety.

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Abdullah et al. (1986) Biotech. 4: 1087.
Almeida and Allshire (2005) TRENDS Cell Biol 15: 251-258.
Alonso et al. (2010) Green Chem. 12: 1493-1513.
Ascherio and Willett (1997) Am. J. Clin. Nutr. 66: 1006S-1010S.
Bartholomew (1971) Temperature effects on the fatty acid composition of developing seeds in safflower, Carthamus tinctorius L.
Baumlein et al. (1991) Mol. Gen. Genet. 225: 459-467.
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Beclin et al. (2002) Current Biology 12: 684-688.
Belide et al. (2011) Plant Methods 7:12.
Bergman et al. (2006) Crop Sci. 46: 1818-1819.
Blacklock et al. (2010) J Biol Chem 285: 28442-28449.
Bligh and Dyer (1959) Canadian Journal of Biochemistry and Physiology 37: 911-7.
Bonanome and Grundy (1988). N. Engl. Med. 318: 1244-1248.
Brodersen and Voinnet (2006) Trends Genet. 22: 268-280.
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Broun et al. (1998) Plant J. 13: 201-210.
Broun et al. (1999) Annu. Rev. Nutr. 19: 197-216.
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Cahoon et al. (2003) Plant J. 34: 671-683.
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Cheng et al. (1996) Plant Cell Rep. 15: 653-657.
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Coutu et al. (2007) Transgenic Research 16: 771-781.
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Falcone et al. (1994) Plant Physiol. 106: 1453-1459.
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Fernandez-Martinez et al. (1993) Euphytica 69: 115-122.
Fofana et al. (2004) Plant Science 166: 1487-1496.
Fujimura et al. (1985) Plant Tissue Culture Lett. 2:74.
Gamez et al. (2003) Food Res International 36: 721-727.
Glevin et al. (2003) Microbiol. Mol. Biol. Rev. 67: 16-37.
Gong and Jiang (2011) Biotechnol. Lett. 33: 1269-1284.
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Gunstone (2001) Inform 11: 1287-1289.
Hafner et al. (2008) Methods 44: 3-12.
Hamdan et al. (2009) Euphytica 168: 61-69.
Henikoff et al. (2004) Plant Physiol 135: 630-636.
Heppard et al. (1996) Plant Physiology 110: 311-319.
Hernandez et al. (2005) Phytochemistry 66: 1417-1426.
Hill and Knowles (1968) Crop Sci. 8: 275-277.
Hinchee et al. (1988) Biotechnology 6: 915-922.
Hu et al. (1997). N. Engl. J. Med. 337: 1491-1499.
Hugly and Somerville (1992) Plant Physiol. 99: 197-202.
Isshiki et al. (2001) Plant Physiol. 125: 1388-1395.
Jofuku et al. (1989) Plant Cell 1: 427-435.
Kang et al. (2011) Plant Physiology and Biochemistry 49: 223-229.
Karmakar et al. (2010) Bioresource Technology 101: 7201-7210.
Khadake et al. (2009) Mol Biotechnol. 42: 168-174.
Kim et al. (2006) Molecular Genetics and Genomics 276: 351-368.
Knothe (2005) Fuel Process. Technol. 86: 1059-1070.
Knothe and Steidley (2005) Fuel 84: 1059-1065
Knowles (1968) Economic Botany 22: 195-200.
Knowles (1969) Economic Botany 23: 324-329.
Knowles (1972) Journal of the American Oil Chemists' Society 49: 27-29.
Knowles (1989) Oil crops of the world: their breeding and utilization / ed G. Robbelen, R. Keith Downey, A. Ashri. McGraw-Hill, New York.
Knowles and Hill (1964) Crop Science 4: 406-409.
Lacroix et al. (2008) Proc. Natl. Acad. Sci. USA 105: 15429-15434.
Langridge et al. (2001) Aust J Agric Res 52: 1043-1077.
Lee et al. (1998) Science 280: 915-918.
Lemieux (2000) Current Genomics 1: 301-311.
Liu (1998) The isolation and characterization of fatty acid desaturase genes in cotton. PhD. Thesis University of Sydney.
Liu et al. (1999) Functional Plant Biology 26: 101-106.
Liu et al. (2001) American Journal of Botany 88: 92-102.
Liu et al. (2002a). J. Am. Coll. Nutr. 21: 205S-211S.
Liu et al. (2002b). Plant Physiol. 129: 1732-1743.
Liu et al. (2010) Fuel 89: 2735-2740.
Loguercio (1998) Current genetics 34: 241-249.
Maher and Bressler (2007) Bioresource Technology 98: 2351-2368.
Marangoni et al. (1995) Trends in Food Sci. Technol. 6: 329-335.
Martinez-Rivas et al. (2001) Molecular Breeding 8: 159-168.
McCartney et al. (2004) Plant J 37: 156-173.
Mensink and Katan (1990). N. Engl. J. Med. 323: 439-445.
Millar and Waterhouse (2005) Funct Integr Genomics 5: 129-135.
Mroczka et al. (2010) Plant Physiology 153: 882-891.
Mundel and Bergman (2009) Safflower. Oil Crops: Handbook of Plant Breeding 4. Ed. J. Vollman and I. Rajcan. Springer Science. Pp 423-447.
Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453.
Niedz et al. (1995) Plant Cell Reports 14: 403.
Noakes and Clifton (1998) Am. J. Clin. Nutr. 98: 242-247.
Nykiforuk et al. (2011) Plant Biotechnology Journal 9: 250-263.
Oil World Annual (2004) International Seed Testing Association (ISTA) Mielke GmbH, Hamburg, Germany
Okuley et al. (1994) Plant Cell 6: 147-158.
Ow et al. (1986) Science 234: 856-859.
Pasquinelli et al. (2005) Curr Opin Genet Develop 15: 200-205.
Paterson et al. (1993) Plant Molecular Biology Reporter 11: 122-127.
Perez-Vich et al. (1998) JAOCS 75: 547-555
Perrin et al. (2000) Mol Breed 6: 345-352.
Peterson et al. (1997) Plant Molecular Biology Reporter 15: 148-153.
Petrie et al. (2010) Plant Methods 6: 8-14.
Phillips et al. (2002) Journal of Food Composition and Analysis 12: 123-142.
Potenza et al. (2004) In Vitro Cell Dev. Biol. Plant 40: 1-22.
Prasher et al. (1985) Biochem. Biophys. Res. Commun. 127: 31-36.
Roche and Gibney (2000) Am. J. Clin. Nutr. 71: 232S-237S.
Ryan et al. (1984) J. Am. Oil Chem. Soc. 61: 1610-1619.
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Claims (18)

Producing seeds that contain triacylglycerols (TAG) consisting of fatty acids esterified to glycerol and / or triacylglycerols (TAG) consisting of fatty acids esterified to glycerol. Can produce plants,
i) the fatty acid comprises palmitic acid and oleic acid;
ii) At least 95% by weight of the oil content of the seed is TAG,
iii) 90% to 95% by weight of the total fatty acids in the oil content of the seed is oleic acid,
iv) Less than 3% by weight of the total fatty acids in the oil content of the seed is palmitic acid,
v) the oil inclusions oleic unsaturated content of less than 0.037 of seed (ODP) and / or palmitic acid of less than 0.063 - linoleic acid - have a oleic acid (PLO) value,
a) a first silencing RNA capable of reducing the expression of a Δ12 desaturase (FAD2) gene in a developing safflower seed as compared to a corresponding safflower seed lacking the first exogenous polynucleotide Wherein the first exogenous polynucleotide is operably linked to a promoter that induces expression of the polynucleotide in the developing safflower seed, and
b) a second capable of reducing the expression of the palmitoyl-ACP thioesterase (FATB) gene in the developing safflower seed as compared to the corresponding safflower seed lacking the second exogenous polynucleotide; A second exogenous polynucleotide encoding a silencing RNA, wherein said second exogenous polynucleotide is operably linked to a promoter that induces expression of said polynucleotide in said developing safflower seed Including safflower seeds.   The safflower seed of claim 1, wherein the oil content of the seed comprises less than 2.25% linoleic acid of total fatty acids. The oil content of the seed has one or more of the following characteristics:
a) 90% to 94%, 91% to 94%, or 91% to 92% by weight of the total fatty acids of the oil content of the seeds is oleic acid,
b) Less than 2.75% by weight or less than 2.5% by weight of the total fatty acids of the oil content of the seed is palmitic acid,
c) 0.1% by weight to 3% by weight or 2% by weight to 3% by weight of the total fatty acids of the oil-containing material of the seeds is polyunsaturated fatty acids (PUFA),
d) less than 1 less than wt%, or 0.5 wt% of the total fatty acids in the oil content of the seed is an α- Reno Len acid (A LA),
e) 90 wt% to 96 wt%, or 92 wt% to 96 wt% of the total fatty acid content of the oil-containing material is a monounsaturated fatty acid,
f) the oil-containing material has an oleic acid monounsaturation (OMP) of less than 0.02, less than 0.015, 0.005 to 0.05, or 0.005 to 0.02.
g) the ODP of the oil content of the seed is 0.01-0.033, 0.016-0.033, or 0.023-0.033, and h) the PLO of the oil content of the seed The value is 0.020 to 0.063, 0.020 to 0.055, 0.020 to 0.050, or 0.050 to 0.055,
The safflower seed of Claim 1 or 2. The oil content of the seed is a lipid further characterized by one or more of the following characteristics:
(A) α-linolenic acid is undetectable in the fatty acids of the lipids,
(B) 55% -80%, 60% -80%, 70% -80%, or at least 60% or at least 70% of the TAG content of the lipid is triolein;
The safflower seed as described in any one of Claims 1-3. A third exogenous that can reduce expression of the plastid ω6 fatty acid desaturase (FAD6) gene in developing safflower seeds as compared to a corresponding safflower seed lacking a third exogenous polynucleotide A polynucleotide, wherein the third exogenous polynucleotide is operably linked to a promoter that induces expression of the polynucleotide in the developing safflower seed;
The safflower seed according to any one of claims 1 to 4, further comprising: The following features a) the first silencing RNA reduces the expression of more than one exogenous gene encoding FAD2 in the developing safflower seed and / or the second silencing RNA is developing Reducing the expression of more than one exogenous gene encoding FATB in safflower seeds in
b) the first exogenous polynucleotide and second exogenous polynucleotide is a single DNA molecule, for connecting between Optionally, the first and the second exogenous polynucleotide DNA Covalently bond with the sequence,
c) the first exogenous polynucleotide and second exogenous polynucleotide is under the control of a single promoter, therefore, said first exogenous polynucleotide and second exogenous poly If the nucleotide is transcribed in safflower seed in the development, the first silencing RNA and the second silencing RN a is covalently linked as part of a single RNA transcript,
d) comprises a single transfer DNA that said seed has integrated into the genome of safflower seeds, said single transfer DNA comprises a first exogenous polynucleotide and second exogenous polynucleotide,
e) the seed is homozygous for the transfer DNA of d)
f) The first silencing RNA and the second silencing RNA are each independently selected from the group consisting of an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, a microRNA, and a double-stranded RNA. To be
g) wherein the promoter is a seed-specific, is selectively expressed in the embryo safflower seeds originating Ikuchu,
h) The seed contains one or more mutants in one or more FAD2 genes, and the mutant is developing as compared to a corresponding safflower seed lacking the mutant Reducing the activity of the one or more FAD2 genes in safflower seeds, wherein the mutation is a deletion, insertion, inversion, frameshift, premature translation stop codon, or one or more non-conservative amino acid substitutions, FAD2 gene Or a null mutant of the CtFAD2-1 gene,
i) FAD2 protein is undetectable in the seed, and j) the first silencing RNA decreases the expression of both CtFAD2-1 and CtFAD2-2 genes,
The safflower seed according to any one of claims 1 to 5, further comprising one or more. 1) The FAD2 gene is one or more of CtFAD2-1 gene, CtFAD2-2 gene, and CtFAD2-10 gene,
2) the FATB gene is a CtFATB-3 gene, and / or 3) the FAD6 gene is a CtFAD6 gene,
Safflower seed according to claim 5 or 6.   A safflower plant capable of producing the seed according to any one of claims 1 to 7. i) obtaining safflower seeds according to any one of claims 1 to 7, and ii) extracting oil from said safflower seeds, thereby producing oil,
A method for producing an oil comprising:   Further comprising purifying the oil extracted from the safflower seed, wherein the purifying step includes degumming, deodorizing, decolorizing, drying, and / or fractionating the extracted oil, and / or the extraction. 10. The method of claim 9, comprising one or more or all of the group consisting of removing at least some, preferably substantially all, waxes and / or wax esters from the treated oil. . Including triacylglycerol (TAG) consisting of fatty acids esterified to glycerol,
i) the fatty acid comprises palmitic acid and oleic acid;
ii) at least 95% by weight of the lipid is TAG;
iii) 90% to 95% by weight of the total fatty acid content of the lipid is oleic acid,
iv) Less than 3% by weight of the total fatty acid content of the lipid is palmitic acid,
v) A safflower seed extract lipid, wherein the lipid has an oleic unsaturation rate (ODP) of less than 0.037 and / or a palmitic-linoleic-oleic acid value (PLO) of less than 0.063. 12. A lipid according to claim 11 comprising linoleic acid in an amount of less than 2.25% by weight of the total fatty acid content. The following features a) 90% to 94%, 91% to 94%, or 91% to 92% by weight of the total fatty acid content of the lipid is oleic acid,
b) Less than 2.75% by weight or less than 2.5% by weight of the total fatty acid content of the lipid is palmitic acid,
c) 0.1 wt% to 3 wt%, or 2 wt% to 3 wt% of the total fatty acid content of the lipid is polyunsaturated fatty acid (PUFA),
d ) Less than 1% or less than 0.5% by weight of the total fatty acid content of the lipid is α-linolenic acid (ALA),
e ) 0.5% to 1% by weight of the total fatty acid content of the lipid is 18: 1Δ11,
f ) The ODP of the fatty acid content of the lipid is 0.01 to 0.033, 0.016 to 0.033, or 0.023 to 0.033.
g ) The PLO value of the fatty acid content of the lipid is 0.020 to 0.063, 0.020 to 0.055, 0.020 to 0.050, or 0.050 to 0.055,
h ) 90% to 96%, or 92% to 96% by weight of the total fatty acid content of the lipid is a monounsaturated fatty acid,
i ) the lipid has an oleic acid monounsaturation ratio (OMP) of less than 0.02, less than 0.015, or 0.005 to 0.02.
j ) the lipid is in the form of a refined oil;
k ) the lipid is not hydrogenated, and
l ) extracted from seed having a volume of at least 1 liter and / or a weight of at least 1 kg and / or obtained from a field-grown plant;
13. A lipid according to claim 11 or 12 , having one or more or all of the following. Further comprising one or more sterols,
In the form of an oil, comprising less than 5 mg sterol / g (oil), or 1.5 mg sterol / g (oil) to 5 mg sterol / g (oil);
a) 2.3% to 4.5% of the total sterol content is ergost-7-en-3β-ol, and b) 1.5% to 3% of the total sterol content is triterpenoid alcohol,
Including one or more of
The lipid according to any one of claims 11 to 13 .  A seed powder extracted from the safflower seed according to any one of claims 1 to 7. A first component, a lipid according to any one of claims 11 to 1 4, and a second component, said second component is non-lipid material, for example, enzymes, non-protein catalyst or chemical A composition that is one or more components of a substance or diet. i) lipids described any one of claims 11 to 1 4, obtaining a safflower seed according to any one of claims 1 6 composition according to or claims 1 to 7,
of ii) Step i), lipids described any one of claims 11 to 1 4, A composition according to claim 1 6, or safflower seed according to any one of claims 1 to 7 Optionally physically processing steps;
ii i) lipids described any one of claims 11 to 1 4, the lipid of the compositions according to claims 1 to 6, safflower seed or steps according to any one of claims 1 to 7 converting at least a portion of the physically treated product of ii) to an industrial product by applying a thermal, chemical, or enzymatic method, or any combination thereof, to the lipid;
iv ) recovering the industrial product ;
Hints, thereby producing industrial products, methods for producing industrial products. i) lipids described any one of claims 11 to 1 4, or lipids of the composition according to claims 1 to 6, or safflower seed according to any one of claims 1 to 7 Reacting with an alcohol, optionally in the presence of a catalyst, to form an alkyl ester, and ii) optionally mixing the alkyl ester with a petroleum fuel;
A method of generating fuel, comprising:

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